RESUMO
PURPOSE: Despite unprecedented responses to immune checkpoint inhibitors and targeted therapy in melanoma, a major subset of patients progresses and have few effective salvage options. We have previously demonstrated robust, selective uptake of the peptidomimetic LLP2A labeled with Cu-64 ([64Cu]-LLP2A) for positron emission tomography (PET) imaging in subcutaneous and metastatic models of B16F10 murine melanoma. LLP2A binds with high affinity to very late antigen-4 (VLA-4, integrin α4ß1), a transmembrane protein overexpressed in melanoma and other cancers that facilitates tumor growth and metastasis. Yet B16F10 fails to faithfully reflect human melanoma biology, as it lacks certain oncogenic driver mutations, including BRAF mutations found in ≥ 50 % of clinical specimens. Here, we evaluated the PET tracer [64Cu]-CB-TE1A1P-PEG4-LLP2A ([64Cu]-LLP2A) in novel, translational BRAFV600E mutant melanoma models differing in VLA-4 expression-BPR (VLA-4-) and BPRα (VLA-4+). PROCEDURES: BPR cells were transduced with α4 (CD49d) to overexpress intact cell surface VLA-4 (BPRα). The binding affinity of [64Cu]-LLP2A to BPR and BPRα cells was determined by saturation binding assays. [64Cu]-LLP2A internalization into B16F10, BPR, and BPRα cells was quantified via a plate-based assay. Tracer biodistribution and PET/CT imaging were evaluated in mice bearing subcutaneous BPR and BPRα tumors. RESULTS: [64Cu]-LLP2A demonstrated high binding affinity to BPRα (Kd = 1.4 nM) but indeterminate binding to BPR cells. VLA-4+ BPRα and B16F10 displayed comparable time-dependent [64Cu]-LLP2A internalization, whereas BPR internalization was undetectable. PET/CT showed increased tracer uptake in BPRα tumors vs. BPR tumors in vivo, which was validated by significantly greater (p < 0.0001) BPRα tumor uptake in biodistribution analyses. CONCLUSIONS: [64Cu]-LLP2A discriminates BPRα (VLA-4+) vs. BPR (VLA-4-) melanomas in vivo, supporting translation of these BRAF-mutated melanoma models via prospective imaging and theranostic studies. These results extend the utility of LLP2A to selectively target clinically relevant and therapy-resistant tumor variants toward its use for therapeutic patient care.
Assuntos
Integrina alfa4beta1 , Melanoma , Animais , Linhagem Celular Tumoral , Radioisótopos de Cobre , Modelos Animais de Doenças , Humanos , Integrina alfa4beta1/metabolismo , Melanoma/diagnóstico por imagem , Melanoma/genética , Camundongos , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada , Tomografia por Emissão de Pósitrons/métodos , Estudos Prospectivos , Proteínas Proto-Oncogênicas B-raf/genética , Distribuição TecidualRESUMO
PURPOSE: Glioblastoma is a lethal brain tumor, heavily infiltrated by tumor-associated myeloid cells (TAMCs). TAMCs are emerging as a promising therapeutic target as they suppress anti-tumor immune responses and promote tumor cell growth. Quantifying TAMCs using non-invasive immunoPET could facilitate patient stratification for TAMC-targeted treatments and monitoring of treatment efficacy. As TAMCs uniformly express the cell surface marker, integrin CD11b, we evaluated a Zr-89 labeled anti-CD11b antibody for non-invasive imaging of TAMCs in a syngeneic orthotopic mouse glioma model. PROCEDURES: A human/mouse cross-reactive anti-CD11b antibody (clone M1/70) was conjugated to a DFO chelator and radiolabeled with Zr-89. PET/CT and biodistribution with or without a blocking dose of anti-CD11b Ab were performed 72 h post-injection (p.i.) of [89Zr]anti-CD11b Ab in mice bearing established orthotopic syngeneic GL261 gliomas and in non tumor-bearing mice. Flow cytometry and immunohistochemistry of dissected GL261 tumors were conducted to confirm the presence of CD11b+ TAMCs. RESULTS: Significant uptake of [89Zr]anti-CD11b Ab was detected at the tumor site (SUVmean = 2.60 ± 0.24) compared with the contralateral hemisphere (SUVmean = 0.6 ± 0.11). Blocking with a 10-fold lower specific activity of [89Zr]anti-CD11b Ab markedly reduced the SUV in the right brain (SUVmean = 0.11 ± 0.06), demonstrating specificity. Spleen and lymph nodes (myeloid cell rich organs) also showed high uptake of the tracer, and biodistribution analysis correlated with the imaging results. CD11b expression within the tumor was validated using flow cytometry and immunohistochemistry, which showed high CD11b expression primarily in the tumoral hemisphere compared with the contralateral hemisphere with very minimal accumulation in non tumor-bearing brain. CONCLUSION: These data establish that [89Zr]anti-CD11b Ab immunoPET targets CD11b+ cells (TAMCs) with high specificity in a mouse model of GBM, demonstrating the potential for non-invasive quantification of tumor-infiltrating CD11b+ immune cells during disease progression and immunotherapy in patients with GBM.
Assuntos
Anticorpos Monoclonais , Neoplasias Encefálicas/diagnóstico por imagem , Antígeno CD11b/imunologia , Glioblastoma/diagnóstico por imagem , Células Mieloides/patologia , Tomografia por Emissão de Pósitrons combinada à Tomografia Computadorizada/métodos , Radioisótopos , Zircônio , Animais , Anticorpos Monoclonais/química , Anticorpos Monoclonais/imunologia , Anticorpos Monoclonais/metabolismo , Anticorpos Monoclonais/farmacocinética , Neoplasias Encefálicas/tratamento farmacológico , Neoplasias Encefálicas/imunologia , Neoplasias Encefálicas/patologia , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Glioblastoma/tratamento farmacológico , Glioblastoma/imunologia , Glioblastoma/patologia , Imunoterapia , Camundongos , Camundongos Endogâmicos C57BL , Células Mieloides/efeitos dos fármacos , Células Mieloides/imunologia , Células Mieloides/metabolismo , Radioisótopos/química , Radioisótopos/farmacocinética , Distribuição Tecidual , Zircônio/química , Zircônio/farmacocinéticaRESUMO
The epithelial sodium channel (ENaC) is expressed in the epithelial cells of the distal convoluted tubules, connecting tubules, and cortical collecting duct (CCD) in the kidney nephron. Under the regulation of the steroid hormone aldosterone, ENaC is a major determinant of sodium (Na+ ) and water balance. The ability of aldosterone to regulate microRNAs (miRs) in the kidney has recently been realized, but the role of miRs in Na+ regulation has not been well established. Here we demonstrate that expression of a miR cluster mmu-miR-23-24-27, is upregulated in the CCD by aldosterone stimulation both in vitro and in vivo. Increasing the expression of these miRs increased Na+ transport in the absence of aldosterone stimulation. Potential miR targets were evaluated and miR-27a/b was verified to bind to the 3'-untranslated region of intersectin-2, a multi-domain protein expressed in the distal kidney nephron and involved in the regulation of membrane trafficking. Expression of Itsn2 mRNA and protein was decreased after aldosterone stimulation. Depletion of Itsn2 expression, mimicking aldosterone regulation, increased ENaC-mediated Na+ transport, while Itsn2 overexpression reduced ENaC's function. These findings reinforce a role for miRs in aldosterone regulation of Na+ transport, and implicate miR-27 in aldosterone's action via a novel target. J. Cell. Physiol. 232: 1306-1317, 2017. © 2016 Wiley Periodicals, Inc.
Assuntos
Aldosterona/farmacologia , MicroRNAs/metabolismo , Néfrons/metabolismo , Sódio/metabolismo , Regulação para Cima/efeitos dos fármacos , Regiões 3' não Traduzidas/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Transporte Biológico/efeitos dos fármacos , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Canais Epiteliais de Sódio/metabolismo , Túbulos Renais Coletores/efeitos dos fármacos , Túbulos Renais Coletores/metabolismo , Camundongos Endogâmicos C57BL , MicroRNAs/genética , Néfrons/efeitos dos fármacos , Ligação Proteica/efeitos dos fármacos , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Ribonuclease III/metabolismo , Transdução de Sinais/efeitos dos fármacosRESUMO
The epithelial sodium channel (ENaC) is the limiting entry point for Na+ reabsorption in the distal kidney nephron and is regulated by numerous hormones, including the mineralocorticoid hormone aldosterone. Previously we identified ankyrin G (AnkG), a cytoskeletal protein involved in vesicular transport, as a novel aldosterone-induced protein that can alter Na+ transport in mouse cortical collecting duct cells. However, the mechanisms underlying AnkG regulation of Na+ transport were unknown. Here we report that AnkG expression directly regulates Na+ transport by altering ENaC activity in the apical membrane. Increasing AnkG expression increased ENaC activity while depleting AnkG reduced ENaC-mediated Na+ transport. These changes were due to a change in ENaC directly rather than through alterations to the Na+ driving force created by Na+/K+-ATPase. Using a constitutively open mutant of ENaC, we demonstrate that the augmentation of Na+ transport is caused predominantly by increasing the number of ENaCs at the surface. To determine the mechanism of AnkG action on ENaC surface number, changes in rates of internalization, recycling, and membrane delivery were investigated. AnkG did not alter ENaC delivery to the membrane from biosynthetic pathways or removal by endocytosis. However, AnkG did alter ENaC insertion from constitutive recycling pathways. These findings provide a mechanism to account for the role of AnkG in the regulation of Na+ transport in the distal kidney nephron.
Assuntos
Anquirinas/metabolismo , Membrana Celular/metabolismo , Canais Epiteliais de Sódio/metabolismo , Túbulos Renais Coletores/metabolismo , ATPase Trocadora de Sódio-Potássio/metabolismo , Animais , Transporte Biológico , Células Cultivadas , Endocitose/fisiologia , Transporte de Íons , Camundongos , RatosRESUMO
A role for microRNAs (miRs) in the physiologic regulation of sodium transport in the kidney has not been established. In this study, we investigated the potential of aldosterone to alter miR expression in mouse cortical collecting duct (mCCD) epithelial cells. Microarray studies demonstrated the regulation of miR expression by aldosterone in both cultured mCCD and isolated primary distal nephron principal cells. Aldosterone regulation of the most significantly downregulated miRs, mmu-miR-335-3p, mmu-miR-290-5p, and mmu-miR-1983 was confirmed by quantitative RT-PCR. Reducing the expression of these miRs separately or in combination increased epithelial sodium channel (ENaC)-mediated sodium transport in mCCD cells, without mineralocorticoid supplementation. Artificially increasing the expression of these miRs by transfection with plasmid precursors or miR mimic constructs blunted aldosterone stimulation of ENaC transport. Using a newly developed computational approach, termed ComiR, we predicted potential gene targets for the aldosterone-regulated miRs and confirmed ankyrin 3 (Ank3) as a novel aldosterone and miR-regulated protein. A dual-luciferase assay demonstrated direct binding of the miRs with the Ank3-3' untranslated region. Overexpression of Ank3 increased and depletion of Ank3 decreased ENaC-mediated sodium transport in mCCD cells. These findings implicate miRs as intermediaries in aldosterone signaling in principal cells of the distal kidney nephron.
Assuntos
Aldosterona/metabolismo , Córtex Renal/metabolismo , Túbulos Renais Coletores/metabolismo , MicroRNAs/metabolismo , Sódio/metabolismo , Aldosterona/genética , Animais , Anquirinas/metabolismo , Transporte Biológico/fisiologia , Linhagem Celular , RNA Helicases DEAD-box/genética , RNA Helicases DEAD-box/metabolismo , Canais Epiteliais de Sódio/metabolismo , Córtex Renal/citologia , Túbulos Renais Coletores/citologia , Luciferases/genética , Camundongos Endogâmicos C57BL , Néfrons/citologia , Néfrons/metabolismo , RNA Interferente Pequeno/genética , Ribonuclease III/genética , Ribonuclease III/metabolismo , Transdução de Sinais/fisiologiaRESUMO
Resveratrol, a naturally occurring phytoalexin, has reported cardioprotective, anti-inflammatory, chemopreventative and antidiabetic properties. Several studies indicate the multiple effects of resveratrol on cellular function are due to its inhibition of class 1A phosphoinositide 3-kinase (PI3K) mediated signaling pathways, but it also activates AMP-activated protein kinase (AMPK). As sodium transport in the kidney via the Epithelial Sodium Channel (ENaC) is highly sensitive to changes in phosphoinositide signaling in the membrane and AMPK, we employed resveratrol to probe the relative effects of phosphatidylinositol species in the plasma membrane and AMPK activity and their impact on ENaC activity in mouse cortical collecting duct (mpkCCDc14) cells. Here we demonstrate that resveratrol acutely reduces amiloride-sensitive current in mpkCCDc14 cells. The time course and dose dependency of this inhibition paralleled depletion of the PI(3,4,5)P3 reporter (AKT-PH) in live-cell microscopy, indicating the early inhibition is likely mediated by resveratrol's known effects on PI3K activity. Additionally, resveratrol induces a late inhibitory effect (4-24 hours) that appears to be mediated via AMPK activation. Resveratrol treatment induces significant AMPK activation compared with vehicle controls after 4 h, which persists through 16 h. Knockdown of AMPK or treatment with the AMPK inhibitor Compound C reduced the late phase of current reduction but had no effect on the early inhibitory activity of resveratrol. Collectively, these data demonstrate that resveratrol inhibits ENaC activity by a dual effect: an early reduction in activity seen within 5 minutes related to depletion of membrane PIP3, and a sustained late (4-24 h) effect secondary to activation of AMPK.
Assuntos
Proteínas Quinases Ativadas por AMP/metabolismo , Canais Epiteliais de Sódio/metabolismo , Túbulos Renais Coletores/citologia , Fosfatidilinositóis/metabolismo , Estilbenos/farmacologia , Proteínas Quinases Ativadas por AMP/genética , Amilorida/farmacologia , Animais , Linhagem Celular , Células Cultivadas , Canais Epiteliais de Sódio/genética , Immunoblotting , Camundongos , RNA Interferente Pequeno , ResveratrolRESUMO
The epithelial sodium channel (ENaC) is the rate-limiting step for sodium reabsorption across tight epithelia. Cyclic-AMP (cAMP) stimulation promotes ENaC trafficking to the apical surface to increase channel number and transcellular Na(+) transport. Removal of corticosteroid supplementation in a cultured cortical collecting duct cell line reduced ENaC expression. Concurrently, the number of vesicles trafficked in response to cAMP stimulation, as measured by a change in membrane capacitance, also decreased. Stimulation with aldosterone restored both the basal and cAMP-stimulated ENaC activity and increased the number of exocytosed vesicles. Knocking down ENaC directly decreased both the cAMP-stimulated short-circuit current and capacitance response in the presence of aldosterone. However, constitutive apical recycling of the Immunoglobulin A receptor was unaffected by alterations in ENaC expression or trafficking. Fischer Rat Thyroid cells, transfected with α,ß,γ-mENaC had a significantly greater membrane capacitance response to cAMP stimulation compared to non-ENaC controls. Finally, immunofluorescent labeling and quantitation revealed a smaller number of vesicles in cells where ENaC expression was reduced. These findings indicate that ENaC is not a passive passenger in regulated epithelial vesicle trafficking, but plays a role in establishing and maintaining the pool of vesicles that respond to cAMP stimulation.
Assuntos
Vesículas Citoplasmáticas/metabolismo , Canais Epiteliais de Sódio/metabolismo , Aldosterona/fisiologia , Animais , Polaridade Celular , Células Cultivadas , Colforsina/farmacologia , Meios de Cultura , AMP Cíclico/fisiologia , Capacitância Elétrica , Células Epiteliais/metabolismo , Células Epiteliais/fisiologia , Canais Epiteliais de Sódio/genética , Expressão Gênica , Técnicas de Silenciamento de Genes , Camundongos , Transporte Proteico , Interferência de RNA , Ratos , Ratos Endogâmicos F344RESUMO
Frequent gene amplification of the receptor-activated calcium-dependent chloride channel TMEM16A (TAOS2 or ANO1) has been reported in several malignancies. However, its involvement in human tumorigenesis has not been previously studied. Here, we show a functional role for TMEM16A in tumor growth. We found TMEM16A overexpression in 80% of head and neck squamous cell carcinoma (SCCHN), which correlated with decreased overall survival in patients with SCCHN. TMEM16A overexpression significantly promoted anchorage-independent growth in vitro, and loss of TMEM16A resulted in inhibition of tumor growth both in vitro and in vivo. Mechanistically, TMEM16A-induced cancer cell proliferation and tumor growth were accompanied by an increase in extracellular signal-regulated kinase (ERK)1/2 activation and cyclin D1 induction. Pharmacologic inhibition of MEK/ERK and genetic inactivation of ERK1/2 (using siRNA and dominant-negative constructs) abrogated the growth effect of TMEM16A, indicating a role for mitogen-activated protein kinase (MAPK) activation in TMEM16A-mediated proliferation. In addition, a developmental small-molecule inhibitor of TMEM16A, T16A-inh01 (A01), abrogated tumor cell proliferation in vitro. Together, our findings provide a mechanistic analysis of the tumorigenic properties of TMEM16A, which represents a potentially novel therapeutic target. The development of small-molecule inhibitors against TMEM16A may be clinically relevant for treatment of human cancers, including SCCHN.
Assuntos
Carcinoma de Células Escamosas/patologia , Transformação Celular Neoplásica , Canais de Cloreto/fisiologia , Neoplasias de Cabeça e Pescoço/patologia , Proteínas Quinases Ativadas por Mitógeno/biossíntese , Proteínas de Neoplasias/fisiologia , Animais , Anoctamina-1 , Carcinoma de Células Escamosas/enzimologia , Carcinoma de Células Escamosas/metabolismo , Divisão Celular , Linhagem Celular , Canais de Cloreto/genética , Canais de Cloreto/metabolismo , Progressão da Doença , Ativação Enzimática , Indução Enzimática , Neoplasias de Cabeça e Pescoço/enzimologia , Neoplasias de Cabeça e Pescoço/metabolismo , Humanos , Hibridização in Situ Fluorescente , Camundongos , Camundongos Knockout , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Fosforilação , Reação em Cadeia da Polimerase Via Transcriptase ReversaRESUMO
Expression of the epithelial sodium channel (ENaC) at the apical membrane of cortical collecting duct (CCD) principal cells is modulated by regulated trafficking mediated by vesicle insertion and retrieval. Small GTPases are known to facilitate vesicle trafficking, recycling, and membrane fusion events; however, little is known about the specific Rab family members that modify ENaC surface density. Using a mouse CCD cell line that endogenously expresses ENaC (mpkCCD), the channel was localized to both Rab11a- and Rab11b-positive endosomes by immunoisolation and confocal fluorescent microscopy. Expression of a dominant negative (DN) form of Rab11a or Rab11b significantly reduced the basal and cAMP-stimulated ENaC-dependent sodium (Na(+)) transport. The greatest reduction in Na(+) transport was observed with the expression of DN-Rab11b. Furthermore, small interfering RNA-mediated knockdown of each Rab11 isoform demonstrated the requirement for Rab11b in ENaC surface expression. These data indicate that Rab11b, and to a lesser extent Rab11a, is involved in establishing the constitutive and cAMP-stimulated Na(+) transport in mpkCCD cells.
Assuntos
Endossomos/metabolismo , Canais Epiteliais de Sódio/metabolismo , Túbulos Renais Coletores/metabolismo , Proteínas rab de Ligação ao GTP/metabolismo , Animais , Linhagem Celular , Células Cultivadas , Células Epiteliais/citologia , Células Epiteliais/metabolismo , Túbulos Renais Coletores/citologia , Camundongos , Transporte ProteicoRESUMO
Alkalosis impairs the natriuretic response to diuretics, but the underlying mechanisms are unclear. The soluble adenylyl cyclase (sAC) is a chemosensor that mediates bicarbonate-dependent elevation of cAMP in intracellular microdomains. We hypothesized that sAC may be an important regulator of Na(+) transport in the kidney. Confocal images of rat kidney revealed specific immunolocalization of sAC in collecting duct cells, and immunoblots confirmed sAC expression in mouse cortical collecting duct (mpkCCD(c14)) cells. These cells exhibit aldosterone-stimulated transepithelial Na(+) currents that depend on both the apical epithelial Na(+) channel (ENaC) and basolateral Na(+),K(+)-ATPase. RNA interference-mediated 60-70% knockdown of sAC expression comparably inhibited basal transepithelial short circuit currents (I(sc)) in mpkCCD(c14) cells. Moreover, the sAC inhibitors KH7 and 2-hydroxyestradiol reduced I(sc) in these cells by 50-60% within 30 min. 8-Bromoadenosine-3',5'-cyclic-monophosphate substantially rescued the KH7 inhibition of transepithelial Na(+) current. Aldosterone doubled ENaC-dependent I(sc) over 4 h, an effect that was abolished in the presence of KH7. The sAC contribution to I(sc) was unaffected with apical membrane nystatin-mediated permeabilization, whereas the sAC-dependent Na(+) current was fully inhibited by basolateral ouabain treatment, suggesting that the Na(+),K(+)-ATPase, rather than ENaC, is the relevant transporter target of sAC. Indeed, neither overexpression of sAC nor treatment with KH7 modulated ENaC currents in Xenopus oocytes. ATPase and biotinylation assays in mpkCCD(c14) cells demonstrated that sAC inhibition decreases catalytic activity rather than surface expression of the Na(+),K(+)-ATPase. In summary, these results suggest that sAC regulates both basal and agonist-stimulated Na(+) reabsorption in the kidney collecting duct, acting to enhance Na(+),K(+)-ATPase activity.
Assuntos
Adenilil Ciclases/metabolismo , Epitélio/metabolismo , Túbulos Renais Coletores/metabolismo , Sódio/metabolismo , Adenosina/análogos & derivados , Adenosina/farmacologia , Inibidores de Adenilil Ciclases , Adenilil Ciclases/genética , Aldosterona/farmacologia , Animais , Biotinilação , Células Cultivadas , Canais Epiteliais de Sódio/metabolismo , Epitélio/efeitos dos fármacos , Estradiol/análogos & derivados , Estradiol/farmacologia , Imunofluorescência , Immunoblotting , Transporte de Íons , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/efeitos dos fármacos , Masculino , Oócitos/citologia , Oócitos/efeitos dos fármacos , Oócitos/metabolismo , RNA Interferente Pequeno/farmacologia , Ratos , Ratos Sprague-Dawley , Bloqueadores dos Canais de Sódio/farmacologia , ATPase Trocadora de Sódio-Potássio/metabolismo , Xenopus laevisRESUMO
The epithelial Na(+) channel (ENaC) is a major regulator of salt and water reabsorption in a number of epithelial tissues. Abnormalities in ENaC function have been directly linked to several human disease states including Liddle's syndrome, psuedohypoaldosteronism, and cystic fibrosis and may be implicated in states as diverse as salt-sensitive hypertension, nephrosis, and pulmonary edema. ENaC activity in epithelial cells is highly regulated both by open probability and number of channels. Open probability is regulated by a number of factors, including proteolytic processing, while ENaC number is regulated by cellular trafficking. This review discusses current understanding of apical membrane delivery, cell surface stability, endocytosis, retrieval, and recycling of ENaC and the molecular partners that have so far been shown to participate in these processes. We review known sites and mechanisms of hormonal regulation of trafficking by aldosterone, vasopressin, and insulin. While many details of the regulation of ENaC trafficking remain to be elucidated, knowledge of these mechanisms may provide further insights into ENaC activity in normal and disease states.
Assuntos
Membrana Celular/fisiologia , Células Epiteliais/fisiologia , Canais Epiteliais de Sódio/fisiologia , Transporte Proteico/fisiologia , Células Epiteliais/citologia , Exocitose/fisiologia , Humanos , Vesículas Transportadoras/fisiologiaRESUMO
We have previously shown that IkappaB kinase-beta (IKKbeta) interacts with the epithelial Na+ channel (ENaC) beta-subunit and enhances ENaC activity by increasing its surface expression in Xenopus oocytes. Here, we show that the IKKbeta-ENaC interaction is physiologically relevant in mouse polarized kidney cortical collecting duct (mpkCCDc14) cells, as RNA interference-mediated knockdown of endogenous IKKbeta in these cells by approximately 50% resulted in a similar reduction in transepithelial ENaC-dependent equivalent short circuit current. Although IKKbeta binds to ENaC, there was no detectable phosphorylation of ENaC subunits by IKKbeta in vitro. Because IKKbeta stimulation of ENaC activity occurs through enhanced channel surface expression and the ubiquitin-protein ligase Nedd4-2 has emerged as a central locus for ENaC regulation at the plasma membrane, we tested the role of Nedd4-2 in this regulation. IKKbeta-dependent phosphorylation of Xenopus Nedd4-2 expressed in HEK-293 cells occurred both in vitro and in vivo, suggesting a potential mechanism for regulation of Nedd4-2 and thus ENaC activity. 32P labeling studies utilizing wild-type or mutant forms of Xenopus Nedd4-2 demonstrated that Ser-444, a key SGK1 and protein kinase A-phosphorylated residue, is also an important IKKbeta phosphorylation target. ENaC stimulation by IKKbeta was preserved in oocytes expressing wild-type Nedd4-2 but blocked in oocytes expressing either a dominant-negative (C938S) or phospho-deficient (S444A) Nedd4-2 mutant, suggesting that Nedd4-2 function and phosphorylation by IKKbeta are required for IKKbeta regulation of ENaC. In summary, these results suggest a novel mode of ENaC regulation that occurs through IKKbeta-dependent Nedd4-2 phosphorylation at a recognized SGK1 and protein kinase A target site.
Assuntos
Canais Epiteliais de Sódio/metabolismo , Regulação da Expressão Gênica/fisiologia , Quinase I-kappa B/metabolismo , Túbulos Renais Coletores/metabolismo , Ubiquitina-Proteína Ligases/metabolismo , Proteínas de Xenopus/metabolismo , Animais , Linhagem Celular , Membrana Celular/genética , Membrana Celular/metabolismo , Polaridade Celular/fisiologia , Complexos Endossomais de Distribuição Requeridos para Transporte , Canais Epiteliais de Sódio/genética , Humanos , Quinase I-kappa B/genética , Proteínas Imediatamente Precoces/genética , Proteínas Imediatamente Precoces/metabolismo , Túbulos Renais Coletores/citologia , Camundongos , Mutação , Ubiquitina-Proteína Ligases Nedd4 , Fosforilação/fisiologia , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Interferência de RNA , Ubiquitina-Proteína Ligases/genética , Proteínas de Xenopus/genética , Xenopus laevisRESUMO
The epithelial sodium channel (ENaC) is ubiquitinated by the E3 ligase Nedd4-2 at the apical membranes of polarized cortical collecting duct (CCD) epithelial cells. This leads to ENaC endocytosis and possible degradation. Because ENaC is known to recycle at the apical membranes of CCD cells, deubiquitinating enzymes (DUBs) are likely involved in regulating ENaC surface density by facilitating ENaC recycling as opposed to degradation. Using a chemical probe approach to tag active DUBs, we identified ubiquitin C-terminal hydrolase (UCH) isoform L3 as the predominant DUB in endosomal compartments of CCD cells. Blocking UCH-L3 activity or reducing its expression by selective knockdown increased ENaC ubiquitination and resulted in its removal from the apical membranes of CCD cells. Functionally this caused a rapid reduction in transepithelial Na(+) currents across the CCD epithelia. Surface biotinylation demonstrated the loss of ENaC from the apical surface when UCH-L3 was inhibited. Whole cell or apical surface immunoprecipitation demonstrated increased ENaC ubiquitination with UCH-L3 inhibition. This constitutes a novel function for UCH in epithelia and in the regulation of ion channels and demonstrates the dynamic regulation of apically located ENaC by recycling, which is facilitated by this DUB.
Assuntos
Cisteína Endopeptidases/fisiologia , Canais Epiteliais de Sódio/metabolismo , Ubiquitina Tiolesterase/fisiologia , Ubiquitina/química , Animais , Biotinilação , Membrana Celular/metabolismo , Clatrina/química , Cisteína Endopeptidases/metabolismo , Endossomos/metabolismo , Células Epiteliais , Túbulos Renais Coletores/metabolismo , Camundongos , Modelos Biológicos , Isoformas de Proteínas , Estrutura Terciária de Proteína , RNA Interferente Pequeno/metabolismo , Fatores de Tempo , Ubiquitina Tiolesterase/metabolismoRESUMO
Ubiquitination of ENaC subunits has been shown to negatively regulate the cell surface expression of ENaC channels. We have previously demonstrated that epsin links ubiquitinated ENaC to clathrin adaptors for clathrin-mediated endocytosis. Epsin is thought to directly modify the curvature of membranes upon binding to phosphatidylinositol 4,5-bisphosphate (PIP2) where it recruits clathrin and stimulates lattice assembly. Murine phosphatidylinositol 4-phosphate 5-kinase alpha (PI5KIalpha) has been shown to enhance endocytosis in a PIP2-dependent manner. We tested the hypothesis that PI5KIalpha-mediated PIP2 production would negatively regulate ENaC current by enhancing epsin-mediated endocytosis of the channel. Expression of PI5KIalpha decreased ENaC currents in Xenopus oocytes by 80%, entirely because of a decrease in cell surface ENaC levels. Catalytically inactive mutants of PI5Kalpha had no effect on ENaC activity. Expression of the PIP2 binding region of epsin increased ENaC current in oocytes, an effect completely reversed by co-expression of PI5KIalpha. Overexpression of epsin reduced amiloride-sensitive current in CCD cells. Overexpression of PI5KIalpha enhanced membrane PIP2 levels and reduced apical surface expression of ENaC in CCD cells, down-regulating amiloride-sensitive current. Knockdown of PI5KIalpha with isoform-specific siRNA resulted in a 4-fold enhancement of ENaC activity. PI5KIalpha localized exclusively to the apical plasma membrane domain when overexpressed in mouse CCD cells, consistent for a role in regulating PIP2 production at the apical plasma membrane. We conclude that membrane turnover events regulating ENaC surface expression and activity in oocytes and CCD cells can be regulated by PI5KIalpha.
Assuntos
Endocitose/fisiologia , Canais Epiteliais de Sódio/biossíntese , Túbulos Renais Coletores/fisiologia , Potenciais da Membrana/fisiologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Amilorida/farmacologia , Animais , Linhagem Celular Transformada , Membrana Celular/enzimologia , Clatrina/genética , Clatrina/metabolismo , Endocitose/efeitos dos fármacos , Canais Epiteliais de Sódio/genética , Expressão Gênica , Isoenzimas/antagonistas & inibidores , Isoenzimas/genética , Isoenzimas/metabolismo , Túbulos Renais Coletores/citologia , Potenciais da Membrana/genética , Camundongos , Antígenos de Histocompatibilidade Menor , Oócitos/citologia , Fosfotransferases (Aceptor do Grupo Álcool)/antagonistas & inibidores , Fosfotransferases (Aceptor do Grupo Álcool)/genética , RNA Interferente Pequeno , Bloqueadores dos Canais de Sódio/farmacologia , Xenopus laevisRESUMO
We previously showed that ENaC is present in lipid rafts in A6 cells, a Xenopus kidney cell line. We now demonstrate that ENaC can be detected in lipid rafts in mouse cortical collecting duct ((MPK)CCD(14)) cells by detergent insolubility, buoyancy on density gradients using two distinct approaches, and colocalization with caveolin 1. Less than 30% of ENaC subunits were found in raft fractions. The channel subunits also colocalized on sucrose gradients with known vesicle targeting and fusion proteins syntaxin 1A, Vamp 2, and SNAP23. Hormonal stimulation of ENaC activity by either forskolin or aldosterone, short or long term, did not alter the lipid raft distribution of ENaC. Methyl-beta-cyclodextrin added apically to (MPK)CCD(14) cells resulted in a slow decline in amiloride-sensitive sodium transport with short circuit current reductions of 38.1 +/- 9.6% after 60 min. The slow decline in ENaC activity in response to apical cyclodextrin was identical to the rate of decline seen when protein synthesis was inhibited by cycloheximide. Apical biotinylation of (MPK)CCD(14) cells confirmed the loss of ENaC at the cell surface following cyclodextrin treatment. Acute stimulation of the recycling pool of ENaC was unaffected by apical cyclodextrin application. Expression of dominant negative caveolin isoforms (CAV1-eGFP and CAV3-DGV) which disrupt caveolae, reduced basal ENaC currents by 72.3 and 78.2%, respectively; but, as with cyclodextrin, the acute response to forskolin was unaffected. We conclude that ENaC is present in and regulated by lipid rafts. The data are consistent with a model in which rafts mediate the constitutive apical delivery of ENaC.
Assuntos
Canais Epiteliais de Sódio/fisiologia , Túbulos Renais Coletores/metabolismo , Microdomínios da Membrana/metabolismo , Animais , Caveolinas/química , Membrana Celular/metabolismo , Células Cultivadas , Colforsina/farmacologia , Ciclodextrinas/química , Cicloeximida/química , Canais Epiteliais de Sódio/química , Genes Dominantes , Proteínas de Fluorescência Verde/química , Camundongos , Modelos Biológicos , Isoformas de Proteínas , Transporte ProteicoRESUMO
Here we present evidence that the epithelial sodium channel (ENaC), a heteromeric membrane protein whose surface expression is regulated by ubiquitination, is present in clathrin-coated vesicles in epithelial cells that natively express ENaC. The channel subunits are ubiquitinated and co-immunoprecipitate with both epsin and clathrin adaptor proteins, and epsin, as expected, co-immunoprecipitates with clathrin adaptor proteins. The functional significance of these interactions was evaluated in a Xenopus oocyte expression system where co-expression of epsin and ENaC resulted in a down-regulation of ENaC activity; conversely, co-expression of epsin sub-domains acted as dominant-negative effectors and stimulated ENaC activity. These results identify epsin as an accessory protein linking ENaC to the clathrin-based endocytic machinery thereby regulating the activity of this ion channel at the cell surface.
Assuntos
Proteínas Adaptadoras de Transporte Vesicular/química , Clatrina/fisiologia , Animais , Membrana Celular/metabolismo , Clatrina/metabolismo , Regulação para Baixo , Eletrofisiologia , Endocitose , Endossomos/metabolismo , Camundongos , Mutação , Oócitos/metabolismo , Estrutura Terciária de Proteína , Ubiquitina/química , XenopusRESUMO
Aldosterone acts to increase apical membrane permeability by activation of epithelial Na(+) channels (ENaC). We have previously shown that aldosterone activates ENaC early in the course of its action by stimulating the methylation of the beta subunit of this heteromeric channel in A6 cells. Aldosterone also stimulates the expression and methylation of k-ras in A6 cells. To determine whether aldosterone-stimulated methylations are seen in mammalian cells, we examined the effect of aldosterone on methylation and ras activation in a continuous line of cultured epithelial cells derived from mouse cortical collecting duct (CCD) and determined that beta mENaC is a substrate for methylation by an enzyme contained in CCD cells. Aldosterone stimulated protein base labile methylation in CCD cells. Aldosterone stimulated Na(+) transport in CCD cells within 1 h of addition and without an increase in cellular amount of any ENaC subunits over the first 4 h. Inhibition of methylation, using the inhibitor 3-deaza-adenosine, blocked the stimulation of Na(+) transport induced by aldosterone at early time points (1-4 h) without affecting cellular amounts of any ENaC subunits. In contrast to 3-deaza-adenosine (3-DZA), which inhibits all methylation reactions, specific inhibitors of small G-protein methylation or prenylation had no effect on the early aldosterone-induced current. Overexpression of isoprenylcysteine carboxylmethyltransferase (PCMTase), the enzyme that methylates ras, had little effect on basal transport but enhanced aldosterone-stimulated transport in A6 cells. Overexpression of PCMTase in CCD cells had no effect on either basal or aldosterone-stimulated transport. Moreover PCMTase had no effect on ENaC activity when co-expressed in Xenopus oocytes. Aldosterone had no effect on either message or protein levels of k-ras in CCD cells. Searching a mouse kidney library, we identified a methyltransferase that stimulates ENaC activity in Xenopus oocytes without affecting surface expression of ENaC. Our results demonstrate that aldosterone stimulates protein methylation in CCD cells, and this is required for expression of the early transport response. In CCD cells this effect is not mediated via methylation of ras, which is not induced by aldosterone in these cells, and the enzyme that methylates ras has no direct effect on ENaC activity. beta ENaC is a substrate for methylation in CCD cells. A novel methyltransferase that stimulates ENaC directly has been identified in CCD cells.
Assuntos
Aldosterona/fisiologia , Canais de Sódio/fisiologia , Animais , Membrana Celular/fisiologia , Células Epiteliais , Canais Epiteliais de Sódio , Túbulos Renais Coletores/citologia , Metilação , Metiltransferases/metabolismo , Camundongos , Oócitos , Proteínas Metiltransferases/biossíntese , Canais de Sódio/biossíntese , Regulação para Cima , Xenopus , Proteínas ras/fisiologiaRESUMO
The epithelial Na(+) channel (ENaC) regulates epithelial salt and water reabsorption, processes that require significant expenditure of cellular energy. To test whether the ubiquitous metabolic sensor AMP-activated kinase (AMPK) regulates ENaC, we examined the effects of AMPK activation on amiloride-sensitive currents in Xenopus oocytes and polarized mouse collecting duct mpkCCD(c14) cells. Microinjection of oocytes expressing mouse ENaC (mENaC) with either active AMPK protein or an AMPK activator inhibited mENaC currents relative to controls as measured by two-electrode voltage-clamp studies. Similarly, pharmacological AMPK activation or overexpression of an activating AMPK mutant in mpkCCD(c14) cells inhibited amiloride-sensitive short circuit currents. Expression of a degenerin mutant beta-mENaC subunit (S518K) along with wild type alpha and gamma increased the channel open probability (P(o)) to approximately 1. However, AMPK activation inhibited currents similarly with expression of either degenerin mutant or wild type mENaC. Single channel recordings under these conditions demonstrated that neither P(o) nor channel conductance was affected by AMPK activation. Moreover, expression of a Liddle's syndrome-type beta-mENaC mutant (Y618A) greatly enhanced ENaC whole cell currents relative to wild type ENaC controls and prevented AMPK-dependent inhibition. These findings indicate that AMPK-dependent ENaC inhibition is mediated through a decrease in the number of active channels at the plasma membrane (N), presumably through enhanced Nedd4-2-dependent ENaC endocytosis. The AMPK-ENaC interaction appears to be indirect; AMPK did not bind ENaC in cells, as assessed by in vivo pull-down assays, nor did it phosphorylate ENaC in vitro. In summary, these results suggest a novel mechanism for coupling ENaC activity and renal Na(+) handling to cellular metabolic status through AMPK, which may help prevent cellular Na(+) loading under hypoxic or ischemic conditions.
Assuntos
Polaridade Celular/fisiologia , Células Epiteliais/enzimologia , Rim/enzimologia , Complexos Multienzimáticos/fisiologia , Proteínas Serina-Treonina Quinases/fisiologia , Canais de Sódio/metabolismo , Proteínas Quinases Ativadas por AMP , Amilorida/farmacologia , Animais , Caspase 2 , Caspases/fisiologia , Linhagem Celular Tumoral , Polaridade Celular/efeitos dos fármacos , Regulação para Baixo/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Ativação Enzimática/fisiologia , Células Epiteliais/efeitos dos fármacos , Canais Epiteliais de Sódio , Feminino , Humanos , Rim/citologia , Rim/efeitos dos fármacos , Camundongos , Oócitos/efeitos dos fármacos , Oócitos/enzimologia , Ratos , Ubiquitina-Proteína Ligases/fisiologia , XenopusRESUMO
Acute hormonal regulation of the epithelial sodium channel (ENaC) in tight epithelia increases transcellular Na(+) transport via trafficking of intracellular channels to the apical surface. The fate of the channels removed from the apical surface following agonist washout is less clear. By repetitively stimulating polarized mouse cortical collecting duct (mCCD, (MPK)CCD(14)) epithelia, we evaluated the hypothesis that ENaC recycles through an intracellular pool to be available for reinsertion into the apical membrane. Short circuit current (I(SC)), membrane capacitance (C(T)), and conductance (G(T)) were recorded from mCCD epithelia mounted in modified Ussing chambers. Surface biotinylation of ENaC demonstrated an increase in channel number in the apical membrane following cAMP stimulation. This increase was accompanied by a 83 +/- 6% (n = 31) increase in I(SC) and a 15.3 +/- 1.5% (n = 15) increase in C(T). Selective membrane permeabilization demonstrated that the C(T) increase was due to an increase in apical membrane capacitance. I(SC) and C(T) declined to basal levels on stimulus washout. Repetitive cAMP stimulation and washout (approximately 1 h each cycle) resulted in response fatigue; DeltaI(SC) decreased approximately 10% per stimulation-recovery cycle. When channel production was blocked by cycloheximide, DeltaI(SC) decreased approximately 15% per stimulation cycle, indicating that newly synthesized ENaC contributed a relatively small fraction of the channels mobilized to the apical membrane. Selective block of surface ENaC by benzamil demonstrated that channels inserted from a subapical pool made up >90% of the stimulated I(SC), and that on restimulation a large proportion of channels retrieved from the apical surface were reinserted into the apical membrane. Channel recycling was disrupted by brefeldin A, which inhibited ENaC exocytosis, by chloroquine, which inhibited ENaC endocytosis and recycling, and by latrunculin A, which blocked ENaC exocytosis. A compartment model featuring channel populations in the apical membrane and intracellular recycling pool provided an adequate kinetic description of the I(SC) responses to repetitive stimulation. The model supports the concept of ENaC recycling in response to repetitive cAMP stimulation.
Assuntos
AMP Cíclico/metabolismo , Exocitose/fisiologia , Ativação do Canal Iônico/fisiologia , Rim/fisiologia , Potenciais da Membrana/fisiologia , Modelos Biológicos , Canais de Sódio/metabolismo , Animais , Linhagem Celular , Colforsina/farmacologia , Simulação por Computador , Canais Epiteliais de Sódio , Exocitose/efeitos dos fármacos , Rim/efeitos dos fármacos , CamundongosRESUMO
Using the yeast two-hybrid system, we identified a number of proteins that interacted with the carboxyl termini of murine epithelial sodium channel (ENaC) subunits. Initial screens indicated an interaction between the carboxyl terminus of beta-ENaC and IkappaB kinase-beta (IKKbeta), the kinase that phosphorylates Ikappabeta and results in nuclear targeting of NF-kappaB. A true two-hybrid reaction employing full-length IKKbeta and the carboxyl termini of all three subunits confirmed a strong interaction with beta-ENaC, a weak interaction with gamma-ENaC, and no interaction with alpha-ENaC. Co-immunoprecipitation studies for IKKbeta were performed in a murine cortical collecting duct cell line that endogenously expresses ENaC. Immunoprecipitation with beta-ENaC, but not gamma-ENaC, resulted in co-immunoprecipitation of IKKbeta. To examine the direct effects of IKKbeta on ENaC activity, co-expression studies were performed using the two-electrode voltage clamp technique in Xenopus oocytes. Oocytes were injected with cRNAs for alphabetagamma-ENaC with or without cRNA for IKKbeta. Co-injection of IKKbeta significantly increased the amiloride-sensitive current above controls. Using cell surface ENaC labeling, we determined that an increase of ENaC in the plasma membrane accounted for the increase in current. The injection of kinase-dead IKKbeta (K44A) in ENaC-expressing oocytes resulted in a significant decrease in current. Treatment of mpkCCD(c14) cells with aldosterone increased whole cell amounts of IKKbeta. Because this result suggested that aldosterone might activate NF-kappaB, mpkCCD(c14) cells were transiently transfected with a luciferase reporter gene responsive to NF-kappaB activation. Both aldosterone and tumor necrosis factor-alpha (TNFalpha) stimulation caused a similar and significant increase in luciferase activity as compared with controls. We conclude that IKKbeta interacts with ENaC by up-regulating ENaC at the plasma membrane and that the presence of IKKbeta is at very least permissive to ENaC function. These studies also suggest a previously unexpected interaction between the NF-kappaB transcription pathway and steroid regulatory pathways in epithelial cells.