RESUMO
Ovarian granulosa cells are essential to gonadotrophin-regulated estrogen production, female cycle maintenance and fertility. The epithelial Na+ channel (ENaC) is associated with female fertility; however, whether and how it plays a role in ovarian cell function(s) remained unexplored. Here, we report patch-clamp and Na+ imaging detection of ENaC expression and channel activity in both human and mouse ovarian granulosa cells, which are promoted by pituitary gonadotrophins, follicle stimulating hormone (FSH) or luteinizing hormone (LH). Cre-recombinase- and CRISPR-Cas9-based granulosa-specific knockout of ENaC α subunit (Scnn1a) in mice resulted in failed estrogen elevation at early estrus, reduced number of corpus luteum, abnormally extended estrus phase, reduced litter size and subfertility in adult female mice. Further analysis using technologies including RNA sequencing and Ca2+ imaging revealed that pharmacological inhibition, shRNA-based knockdown or the knockout of ENaC diminished spontaneous or stimulated Ca2+ oscillations, lowered the capacity of intracellular Ca2+ stores and impaired FSH/LH-stimulated transcriptome changes for estrogen production in mouse and/or human granulosa cells. Together, these results have revealed a previously undefined role of ENaC in modulating gonadotrophin signaling in granulosa cells for estrogen homeostasis and thus female fertility.
Assuntos
Cálcio , Canais Epiteliais de Sódio , Estrogênios , Fertilidade , Células da Granulosa , Homeostase , Feminino , Animais , Células da Granulosa/metabolismo , Canais Epiteliais de Sódio/metabolismo , Canais Epiteliais de Sódio/genética , Humanos , Estrogênios/metabolismo , Camundongos , Fertilidade/genética , Cálcio/metabolismo , Gonadotropinas/metabolismo , Transdução de Sinais , Camundongos Knockout , Sinalização do CálcioRESUMO
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.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística , Neurônios , Feminino , Animais , Camundongos , Ratos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Encéfalo , Diestro , EletricidadeRESUMO
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.
Assuntos
Diferenciação Celular/efeitos dos fármacos , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Desenvolvimento Embrionário/efeitos dos fármacos , Células-Tronco/citologia , Células-Tronco/metabolismo , Transcrição Gênica/efeitos dos fármacos , Tretinoína/farmacologia , Xenopus laevis/embriologia , Animais , Sequência de Bases , Linhagem Celular , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/efeitos dos fármacos , Humanos , Masculino , Camundongos Endogâmicos C57BL , Receptor alfa de Ácido Retinoico/metabolismo , Transdução de Sinais/efeitos dos fármacos , Espermatogônias/citologia , Células-Tronco/efeitos dos fármacos , Xenopus laevis/metabolismo , beta Catenina/metabolismoRESUMO
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.
Assuntos
Histona Desmetilases/genética , MicroRNAs/genética , Ovário/crescimento & desenvolvimento , Síndrome de Turner/genética , Inativação do Cromossomo X/genética , Adolescente , Adulto , Sequência de Aminoácidos , Linhagem Celular Tumoral , Imunoprecipitação da Cromatina , Regulação para Baixo , Feminino , Regulação da Expressão Gênica , Ontologia Genética , Células HEK293 , Humanos , Leucócitos Mononucleares/metabolismo , MicroRNAs/sangue , Regiões Promotoras Genéticas , Análise de Sequência de RNA , Regulação para Cima , Adulto JovemRESUMO
Spermatogenesis is a multistep process that supports the production of millions of sperm daily. Understanding of the molecular mechanisms that regulate spermatogenesis has been a major focus for decades. Yet, the regulators involved in different cellular processes of spermatogenesis remain largely unknown. Human diseases that result in defective spermatogenesis have provided hints on the molecular mechanisms regulating this process. In this review, we have summarized recent findings on the function and signaling mechanisms of several genes that are known to be associated with disease or pathological processes, including CFTR, CD147, YWK-II and CT genes, and discuss their potential roles in regulating different processes of spermatogenesis.
Assuntos
Células de Sertoli/fisiologia , Espermatogênese/fisiologia , Espermatozoides/citologia , Precursor de Proteína beta-Amiloide/genética , Precursor de Proteína beta-Amiloide/metabolismo , Basigina/genética , Basigina/metabolismo , Movimento Celular/genética , Criptorquidismo/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Humanos , Masculino , Proteínas de Membrana/genética , Proteínas de Membrana/metabolismo , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Células de Sertoli/citologia , Transdução de SinaisRESUMO
Mutations in CFTR lead to dysfunction of tubular organs, which is currently attributed to impairment of its conductive properties. We now show that CFTR regulates tight junction assembly and epithelial cell differentiation through modulation of the ZO-1-ZONAB pathway. CFTR colocalizes with ZO-1 at the tight junctions of trachea and epididymis, and is expressed before ZO-1 in Wolffian ducts. CFTR interacts with ZO-1 through the CTFR PDZ-binding domain. In a three-dimensional (3D) epithelial cell culture model, CFTR regulates tight junction assembly and is required for tubulogenesis. CFTR inhibition or knockdown reduces ZO-1 expression and induces the translocation of the transcription factor ZONAB (also known as YBX3) from tight junctions to the nucleus, followed by upregulation of the transcription of CCND1 and downregulation of ErbB2 transcription. The epididymal tubules of cftr(-/-) and cftr(ΔF508) mice have reduced ZO-1 levels, increased ZONAB nuclear expression, and decreased epithelial cell differentiation, illustrated by the reduced expression of apical AQP9 and V-ATPase. This study provides a new paradigm for the etiology of diseases associated with CFTR mutations, including cystic fibrosis.
Assuntos
Núcleo Celular/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Fibrose Cística/metabolismo , Proteínas de Ligação a DNA/metabolismo , Células Epiteliais/fisiologia , Junções Íntimas/fisiologia , Fatores de Transcrição/metabolismo , Proteína da Zônula de Oclusão-1/metabolismo , Animais , Diferenciação Celular/genética , Células Cultivadas , Ciclina D1/genética , Ciclina D1/metabolismo , Fibrose Cística/genética , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Proteínas de Ligação a DNA/genética , Regulação da Expressão Gênica/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Organogênese/genética , Ligação Proteica , Transporte Proteico/genética , Receptor ErbB-2/genética , Receptor ErbB-2/metabolismo , Transdução de Sinais/genética , Fatores de Transcrição/genética , Proteína da Zônula de Oclusão-1/genéticaRESUMO
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.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Epiderme/metabolismo , Epiderme/patologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , NF-kappa B/metabolismo , Cicatrização , Animais , Diferenciação Celular , Proliferação de Células , Regulador de Condutância Transmembrana em Fibrose Cística/deficiência , Humanos , Inflamação/patologia , Queratinócitos/metabolismo , Queratinócitos/patologia , Masculino , Camundongos Transgênicos , Transdução de SinaisRESUMO
Women with polycystic ovary syndrome (PCOS) undergoing IVF-embryo transfer based-assisted reproductive technology (ART) treatment show variable ovarian responses to exogenous FSH administration. For better understanding and control of PCOS ovarian responses in ART, the present study was carried out to compare the follicular hormones and the expression of granulosa cell genes between PCOS and non-PCOS women during ART treatment as well as their IVF outcomes. Overall, 138 PCOS and 78 non-PCOS women were recruited for the present study. Follicular fluid collected from PCOS women showed high levels of testosterone. The expression of aromatase was found significantly reduced in luteinized granulosa cells from PCOS women. In cultured luteinized granulosa cells isolated from non-PCOS women, their exposure to testosterone at a level that was observed in PCOS follicles could decrease both mRNA and protein levels of aromatase in vitro. The inhibitory effect of testosterone was abolished by androgen receptor antagonist, flutamide. These results suggest that the hyperandrogenic follicular environment may be a key hazardous factor leading to the down-regulation of aromatase in PCOS.
Assuntos
Aromatase/metabolismo , Hiperandrogenismo/metabolismo , Células Lúteas/metabolismo , Luteína/metabolismo , Síndrome do Ovário Policístico/metabolismo , Adulto , Antagonistas de Androgênios/farmacologia , Linhagem Celular , Regulação para Baixo/genética , Feminino , Fertilização in vitro , Flutamida/farmacologia , Líquido Folicular/metabolismo , Humanos , Gravidez , RNA Mensageiro/biossíntese , Técnicas de Reprodução Assistida , Testosterona/metabolismo , Resultado do TratamentoRESUMO
In our previous study, we have demonstrated that the epithelial sodium channel (ENaC) mediates the embryo-derived signals leading to the activation of CREB and upregulation of cyclooxygenase type 2 (COX2) required for embryo implantation. This study aims to investigate whether microRNAs (miRNAs) are involved in the ENaC-induced upregulation of COX2 during embryo implantation. The results show that the levels of miR-101 and miR-199a-3p, two COX2 targeting miRNAs, are reduced by ENaC activation, and increased by ENaC inhibition or knock-down of ENaC subunit (ENaCα) in human endometrial surface epithelial (HES) cells or in mouse uteri during implantation. Phosphorylation of CREB is induced by the activation of ENaC, and blocked by ENaC inhibition or knockdown in HES cells. Knockdown of ENaCα or CREB in HES cells or in mouse uterus in vivo results in increases in miR-101 and miR-199a-3p, accompanied with decreases in COX2 protein levels and reduction in implantation rate. The downregulation of COX2 caused by knockdown of ENaC or CREB can be recovered by the inhibitors of miR-101 or miR-199a-3p in HES cells. These results reveal a novel molecular mechanism modulating COX2 expression during embryo implantation via ENaC-dependent CREB activation and COX2-targeting miRNAs.
Assuntos
Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/metabolismo , Implantação do Embrião/fisiologia , Canais Epiteliais de Sódio/fisiologia , MicroRNAs/fisiologia , Animais , Células Cultivadas , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/efeitos dos fármacos , Ciclo-Oxigenase 2/fisiologia , Embrião de Mamíferos/fisiologia , Endométrio/citologia , Células Epiteliais/citologia , Feminino , Humanos , Técnicas In Vitro , Camundongos , Camundongos Endogâmicos ICR , Modelos Animais , Fosforilação/fisiologia , RNA Interferente Pequeno/farmacologia , Transdução de Sinais/fisiologiaRESUMO
Although microRNAs (miRNAs) have been recognized as one of the important epigenetic mechanisms that regulate gene expression in response to changes in the environment, the links between environmental cues and changes in miRNAs remain largely unknown. Localized to the cell membrane and recognized as an anion channel, the cystic fibrosis transmembrane conductance regulator (CFTR) has recently been shown to mediate important signalling pathways leading to activation of miRNAs. This brief review summarizes the related findings and discusses the emerging role of CFTR as an epigenetic regulator, possibly involved in a wide spectrum of physiological and pathological processes.
Assuntos
Regulador de Condutância Transmembrana em Fibrose Cística/genética , Epigênese Genética/genética , Regulação da Expressão Gênica/genética , MicroRNAs/genética , Membrana Celular/fisiologia , Sinais (Psicologia) , Regulador de Condutância Transmembrana em Fibrose Cística/fisiologia , Meio Ambiente , Regulação da Expressão Gênica/fisiologia , Humanos , MicroRNAs/fisiologia , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
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.
Assuntos
Hiperinsulinismo , Resistência à Insulina , Síndrome do Ovário Policístico , Camundongos , Feminino , Ratos , Humanos , Animais , Adulto , Síndrome do Ovário Policístico/metabolismo , Androgênios/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Flutamida/farmacologia , Regulação para Cima , Resistência à Insulina/fisiologia , Insulina , Di-Hidrotestosterona/farmacologia , Glucose/farmacologiaRESUMO
Magnesium (Mg) has gained widespread recognition as a potential revolutionary orthopedic biomaterial. However, whether the biodegradation of the Mg-based orthopedic implants would pose a risk to patients with chronic kidney disease (CKD) remains undetermined as the kidney is a key organ regulating mineral homeostasis. A rat CKD model was established by a 5/6 subtotal nephrectomy approach, followed by intramedullary implantation of three types of pins: stainless steel, high pure Mg with high corrosion resistance, and the Mg-Sr-Zn alloy with a fast degradation rate. The long-term biosafety of the biodegradable Mg or its alloys as orthopedic implants were systematically evaluated. During an experimental period of 12 weeks, the implantation did not result in a substantial rise of Mg ion concentration in serum or major organs such as hearts, livers, spleens, lungs, or kidneys. No pathological changes were observed in organs using various histological techniques. No significantly increased iNOS-positive cells or apoptotic cells in these organs were identified. The biodegradable Mg or its alloys as orthopedic implants did not pose an extra health risk to CKD rats at long-term follow-up, suggesting that these biodegradable orthopedic devices might be suitable for most target populations, including patients with CKD.
Assuntos
Implantes Absorvíveis , Ligas , Magnésio , Insuficiência Renal Crônica , Animais , Magnésio/química , Ligas/química , Ratos , Insuficiência Renal Crônica/terapia , Insuficiência Renal Crônica/metabolismo , Masculino , Ratos Sprague-Dawley , Materiais Biocompatíveis/química , Teste de Materiais , Rim/metabolismo , Rim/patologia , Aço Inoxidável/química , CorrosãoRESUMO
Clear cells express the vacuolar proton-pumping H(+)-ATPase (V-ATPase) and acidify the lumen of the epididymis, a process that is essential for male fertility. The renin-angiotensin-aldosterone system (RAAS) regulates fluid and electrolyte balance in the epididymis, and a previous study showed binding of aldosterone exclusively to epididymal clear cells (Hinton BT, Keefer DA. Steroid Biochem 23: 231-233, 1985). We examined here the role of aldosterone in the regulation of V-ATPase in the epididymis. RT-PCR showed expression of the mineralocorticoid receptor [MR; nuclear receptor subfamily 3, group C member 2 (NR3C2)] and 11-ß-dehydrogenase isozyme 2 (HSD11ß2) mRNAs specifically in clear cells, isolated by fluorescence-activated cell sorting from B1-enhanced green fluorescent protein (EGFP) mice. Tail vein injection of adult rats with aldosterone, 1,2-dioctanoyl-sn-glycerol (DOG), or 8-(4-chlorophenylthio)-cAMP (cpt-cAMP) induced V-ATPase apical membrane accumulation and extension of V-ATPase-labeled microvilli in clear cells in the caput epididymis but not in the cauda. V-ATPase activity was measured in EGFP-expressing clear cells using the intracellular pH (pHi)-sensing dye seminaphthorhodafluor-5F-5-(and 6)-carboxylic acid, acetoxymethyl ester acetate (SNARF-5F). Aldosterone induced a rapid increase in the rate of Na(+)- and bicarbonate-independent pHi recovery following an NH4Cl-induced acid load in clear cells isolated from the caput but not the cauda. This effect was abolished by concanamycin A, spironolactone, and chelerythrine but not myristoylated-protein kinase inhibitor (mPKI) or mifepristone. Thus aldosterone increases V-ATPase-dependent proton secretion in clear cells in the caput epididymis via MR/NR3C2 and PKC activation. This study, therefore, identifies aldosterone as an active member of the RAAS for the regulation of luminal acidification in the proximal epididymis.
Assuntos
Aldosterona/metabolismo , Epididimo/enzimologia , Sistema Renina-Angiotensina , ATPases Vacuolares Próton-Translocadoras/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/genética , 11-beta-Hidroxiesteroide Desidrogenase Tipo 2/metabolismo , Animais , Separação Celular/métodos , Epididimo/citologia , Epididimo/efeitos dos fármacos , Citometria de Fluxo , Proteínas de Fluorescência Verde/genética , Proteínas de Fluorescência Verde/metabolismo , Concentração de Íons de Hidrogênio , Masculino , Camundongos , Camundongos Transgênicos , Proteína Quinase C/metabolismo , RNA Mensageiro/metabolismo , Ratos , Ratos Sprague-Dawley , Receptores de Mineralocorticoides/genética , Receptores de Mineralocorticoides/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Sistema Renina-Angiotensina/efeitos dos fármacos , Transdução de Sinais , Fatores de Tempo , Regulação para Cima , ATPases Vacuolares Próton-Translocadoras/genéticaRESUMO
Changes that occur to mammalian sperm upon epididymal transit and maturation render these cells capable of moving progressively and capacitating. Signaling events leading to mammalian sperm capacitation depend on the modulation of proteins by phosphorylation and dephosphorylation cascades. Recent experiments have demonstrated that the Src family of kinases plays an important role in the regulation of these events. However, sperm from cSrc null mice display normal tyrosine phosphorylation associated with capacitation. We report here that, despite normal phosphorylation, sperm from cSrc null mice display a severe reduction in forward motility, and are unable to fertilize in vitro. Histological analysis of seminiferous tubules in the testes, caput and corpus epididymis do not reveal obvious defects. However, the cauda epididymis is significantly smaller, and expression of key transport proteins in the epithelial cells lining this region is reduced in cSrc null mice compared to wild type littermates. Although previously, we and others have shown the presence of cSrc in mature sperm from cauda epididymis, a closer evaluation indicates that this tyrosine kinase is not present in sperm from the caput epididymis, suggesting that this protein is acquired by sperm later during epididymal maturation. Consistent with this observation, cSrc is enriched in vesicles released by the epididymal epithelium known as epididymosomes. Altogether, these observations indicate that cSrc is essential for cauda epididymal development and suggest an essential role of this kinase in epididymal sperm maturation involving cSrc extracellular trafficking.
Assuntos
Epididimo/crescimento & desenvolvimento , Epididimo/metabolismo , Espermatozoides/metabolismo , Animais , Epididimo/citologia , Regulação da Expressão Gênica no Desenvolvimento , Processamento de Imagem Assistida por Computador , Masculino , Camundongos , Camundongos Knockout , Tamanho do Órgão , Proteínas Proto-Oncogênicas pp60(c-src)/genética , Proteínas Proto-Oncogênicas pp60(c-src)/metabolismo , Capacitação Espermática/fisiologia , Motilidade dos Espermatozoides/fisiologia , Espermatozoides/citologiaRESUMO
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.
Assuntos
Adenocarcinoma de Pulmão , Neoplasias Pulmonares , Humanos , Glutationa S-Transferase pi/genética , Glutationa S-Transferase pi/metabolismo , Fator 2 Relacionado a NF-E2 , Neoplasias Pulmonares/tratamento farmacológico , Neoplasias Pulmonares/metabolismo , Adenocarcinoma de Pulmão/tratamento farmacológico , Receptores Proteína Tirosina QuinasesRESUMO
Extracellular ATP is essential for the function of the epididymis and spermatozoa, but ATP release in the epididymis remains uncharacterized. We investigated here whether epithelial cells release ATP into the lumen of the epididymis, and we examined the role of the cystic fibrosis transmembrane conductance regulator (CFTR), a Cl(-) and HCO(3)(-) conducting ion channel known to be associated with male fertility, in this process. Immunofluorescence labelling of mouse cauda epididymidis showed expression of CFTR in principal cells but not in other epithelial cells. CFTR mRNA was not detectable in clear cells isolated by fluorescence-activated cell sorting (FACS) from B1-EGFP mice, which express enhanced green fluorescent protein (EGFP) exclusively in these cells in the epididymis. ATP release was detected from the mouse epididymal principal cell line (DC2) and increased by adrenaline and forskolin. Inhibition of CFTR with CFTR(inh172) and transfection with CFTR-specific siRNAs in DC2 cells reduced basal and forskolin-activated ATP release. CFTR-dependent ATP release was also observed in primary cultures of mouse epididymal epithelial cells. In addition, steady-state ATP release was detected in vivo in mice, by measuring ATP concentration in a solution perfused through the lumen of the cauda epididymidis tubule and collected by cannulation of the vas deferens. Luminal CFTR(inh172) reduced the ATP concentration detected in the perfusate. This study shows that CFTR is involved in the regulation of ATP release from principal cells in the cauda epididymidis. Given that mutations in CFTR are a leading cause of male infertility, we propose that defective ATP signalling in the epididymis might contribute to dysfunction of the male reproductive tract associated with these mutations.
Assuntos
Trifosfato de Adenosina/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Epididimo/metabolismo , Animais , Sequência de Bases , Linhagem Celular , Colforsina/farmacologia , Regulador de Condutância Transmembrana em Fibrose Cística/antagonistas & inibidores , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Epididimo/citologia , Epididimo/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Técnicas de Silenciamento de Genes , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , RNA Interferente Pequeno/genética , Transdução de SinaisRESUMO
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.
Assuntos
Bicarbonatos/metabolismo , Regulador de Condutância Transmembrana em Fibrose Cística/metabolismo , Linfócitos/fisiologia , Animais , Anidrases Carbônicas/metabolismo , Linhagem Celular , Chlamydia trachomatis/imunologia , Técnicas de Cocultura , Regulador de Condutância Transmembrana em Fibrose Cística/genética , Humanos , Concentração de Íons de Hidrogênio , Linfócitos/citologia , Camundongos , Camundongos Endogâmicos CFTR , Camundongos Knockout , Pseudomonas aeruginosa/imunologia , Mucosa Respiratória/citologia , Mucosa Respiratória/metabolismoRESUMO
As an analog to the endothelium situated next to the vascular smooth muscle, the epithelium is emerging as an important regulator of smooth muscle contraction in many vital organs/tissues by interacting with other cell types and releasing epithelium-derived factors, among which prostaglandins have been demonstrated to play a versatile role in governing smooth muscle contraction essential to the physiological and pathophysiological processes in a wide range of organ systems.
Assuntos
Células Epiteliais/metabolismo , Epitélio/metabolismo , Contração Muscular , Músculo Liso/metabolismo , Miócitos de Músculo Liso/metabolismo , Comunicação Parácrina , Prostaglandinas/metabolismo , Animais , Humanos , Transdução de SinaisRESUMO
In the earlier study, sodium orthovanadate (SOV) has been reported to be a powerful inhibitor of (Na(+), K(+)) adenosine triphosphatase, exhibit widespread actions on the renal and cardiovascular systems, induces smooth muscle contraction by inhibiting the phosphorylation of the protein tyrosine phosphatases. In the current study, we aimed to investigate the cellular mechanisms by which SOV facilitated contractile response of vas deferens smooth muscle and its potential therapeutic advantage. Exogenous application of ATP and NA-caused contraction was strengthened by pretreatment with SOV. This facilitation was inhibited not by bath with the inhibitor of P2 receptor, PPADS, or the inhibitor of α1 receptor, Prazosin, but by bath with the protein tyrosine kinase inhibitor, Genistein. SOV induced a sustained increase in intracellular Ca(2+) of smooth muscle cells, which was abolished by 100 µM Genistein or Ca(2+)-free solution. The facilitation of SOV could also be inhibited by the selective inhibitors of TRP channel, 2-APB and non-selective cation channel, Gd(3+), Ni(+). The in vivo study showed that peritoneal injection of SOV in dystrophic mice (mdx mice) enhanced the contraction of vas deferens smooth muscle stimulated by electrical field stimulation, ATP, noradrenaline, or KCl. The above results suggest that SOV facilitates the concentration of vas deferens smooth muscle through the tyrosine phosphorylation activated the non-selective cation channels, which has potential use in the therapy for muscle dysfunction.
Assuntos
Contração Muscular/efeitos dos fármacos , Músculo Liso/efeitos dos fármacos , Vanadatos/farmacologia , Ducto Deferente/fisiologia , Trifosfato de Adenosina/farmacologia , Antagonistas de Receptores Adrenérgicos alfa 1 , Agonistas alfa-Adrenérgicos/farmacologia , Animais , Compostos de Boro/farmacologia , Sinalização do Cálcio/efeitos dos fármacos , Células Cultivadas , Interações Medicamentosas , Estimulação Elétrica , Genisteína/farmacologia , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos mdx , Músculo Liso/fisiologia , Norepinefrina/farmacologia , Técnicas de Patch-Clamp , Prazosina/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Ratos , Ratos Sprague-Dawley , Canais de Potencial de Receptor Transitório/agonistas , Canais de Potencial de Receptor Transitório/antagonistas & inibidores , Ducto Deferente/citologiaRESUMO
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 (â¼23%) 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 (â¼50%) 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.