274 results on '"Guggino WB"'
Search Results
2. Low-molecular weight protein handling is largely preserved in cystic fibrosis (CF) kidney
- Author
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UCL - Cliniques universitaires Saint-Luc, UCL - MD/MINT - Département de médecine interne, UCL - MD/BICL - Département de biochimie et de biologie cellulaire, Devuyst, Olivier, Jouret, François, Dom, Geneviève, Guggino, WB, Cassiman, JJ., de Jonge, HR, Courtoy, Pierre J., 36th Annual Meeting of the American-Society-of-Nephrology, UCL - Cliniques universitaires Saint-Luc, UCL - MD/MINT - Département de médecine interne, UCL - MD/BICL - Département de biochimie et de biologie cellulaire, Devuyst, Olivier, Jouret, François, Dom, Geneviève, Guggino, WB, Cassiman, JJ., de Jonge, HR, Courtoy, Pierre J., and 36th Annual Meeting of the American-Society-of-Nephrology
- Published
- 2003
3. ClC5 knock-out induces multiple alterations in the endocytic apparatus of proximal tubular cells.
- Author
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UCL - Cliniques universitaires Saint-Luc, UCL - MD/MINT - Département de médecine interne, UCL - MD/BICL - Département de biochimie et de biologie cellulaire, Christensen, EI, Devuyst, Olivier, Dom, Geneviève, Nielsen, R, Leruth, Michèle, Guggino, WB, Courtoy, Pierre J., UCL - Cliniques universitaires Saint-Luc, UCL - MD/MINT - Département de médecine interne, UCL - MD/BICL - Département de biochimie et de biologie cellulaire, Christensen, EI, Devuyst, Olivier, Dom, Geneviève, Nielsen, R, Leruth, Michèle, Guggino, WB, and Courtoy, Pierre J.
- Published
- 2002
4. CIC-5 is an essential component of the endoytic machinery in proximal tubule cells
- Author
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UCL - Cliniques universitaires Saint-Luc, UCL - MD/MINT - Département de médecine interne, Hryeiw, DH, Devuyst, Olivier, Guggino, WB, UCL - Cliniques universitaires Saint-Luc, UCL - MD/MINT - Département de médecine interne, Hryeiw, DH, Devuyst, Olivier, and Guggino, WB
- Published
- 2002
5. Regulation of receptor mediated endocytosis by cofilin in LLC-PK1 cells.
- Author
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UCL - Cliniques universitaires Saint-Luc, UCL - MD/MINT - Département de médecine interne, Hryciw, D, Devuyst, Olivier, Wang, YH, Guggino, WB, UCL - Cliniques universitaires Saint-Luc, UCL - MD/MINT - Département de médecine interne, Hryciw, D, Devuyst, Olivier, Wang, YH, and Guggino, WB
- Published
- 2002
6. Mice lacking renal chloride channel, CLC-5, are a model for Dent's disease, a nephrolithiasis disorder associated with defective receptor-mediated endocytosis
- Author
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UCL - Cliniques universitaires Saint-Luc, UCL - MD/MINT - Département de médecine interne, Wang, SS, Devuyst, Olivier, Courtoy, Pierre J., Wang, XT, Wang, H, Wang, YS, Thakker, RV, Guggino, S, Guggino, WB, UCL - Cliniques universitaires Saint-Luc, UCL - MD/MINT - Département de médecine interne, Wang, SS, Devuyst, Olivier, Courtoy, Pierre J., Wang, XT, Wang, H, Wang, YS, Thakker, RV, Guggino, S, and Guggino, WB
- Abstract
Nephrolithiasis (kidney stones) affects 5-10% of adults and is most commonly associated with hypercalciuria, which may be due to monogenic renal tubular disorders. One such hypercalciuric disorder is Dent's disease, which is characterized by renal proximal tubular defects that include low molecular weight proteinuria, aminoaciduria and glycosuria, together with rickets in some patients. Dent's disease is due to inactivating mutations of the renal-specific voltage-gated chloride channel, CLC-5, which is expressed in the proximal tubule, thick ascending limb and collecting duct. The subcellular localization of CLC-5 to the proximal tubular endosomes has suggested a role in endocytosis, and to facilitate in vivo investigations of CLC-5 in Dent's disease we generated mice lacking CLC-5 by targeted gene disruption. CLC-5-deficient mice developed renal tubular defects which included low molecular weight (<70 kDa) proteinuria, generalized aminoaciduria that was more pronounced for neutral and polar amino acids, and glycosuria. They also developed hypercalciuria and renal calcium deposits and some had deformities of the spine. Furthermore, endocytosis as assessed by horseradish peroxidase uptake in the proximal tubule was severely impaired in CLC-5-deficient mice, thereby demonstrating a role for CLC-5 in endosomal uptake of low molecular weight proteins. Thus, CLC-5-deficient mice provide a model for Dent's disease and this will help in elucidating the function of this chloride channel in endocytosis and renal calcium homeostasis.
- Published
- 2000
7. Thyroid hormone modulates ClC-2 chloride channel gene expression in rat renal proximal tubules
- Author
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Santos Ornellas, D, primary, Grozovsky, R, additional, Goldenberg, RC, additional, Carvalho, DP, additional, Fong, P, additional, Guggino, WB, additional, and Morales, M, additional
- Published
- 2003
- Full Text
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8. A single histidine residue is essential for zinc inhibition of GABA rho 1 receptors
- Author
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Wang, TL, primary, Hackam, A, additional, Guggino, WB, additional, and Cutting, GR, additional
- Published
- 1995
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9. A novel gamma-aminobutyric acid receptor subunit (rho 2) cloned from human retina forms bicuculline-insensitive homooligomeric receptors in Xenopus oocytes
- Author
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Wang, TL, primary, Guggino, WB, additional, and Cutting, GR, additional
- Published
- 1994
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10. Future directions in early cystic fibrosis lung disease research: an NHLBI workshop report.
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Ramsey BW, Banks-Schlegel S, Accurso FJ, Boucher RC, Cutting GR, Engelhardt JF, Guggino WB, Karp CL, Knowles MR, Kolls JK, Lipuma JJ, Lynch S, McCray PB Jr, Rubenstein RC, Singh PK, Sorscher E, Welsh M, Ramsey, Bonnie W, Banks-Schlegel, Susan, and Accurso, Frank J
- Abstract
Since the 1989 discovery that mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), there has been substantial progress toward understanding the molecular basis for CF lung disease, leading to the discovery and development of new therapeutic approaches. However, the earliest impact of the loss of CFTR function on airway physiology and structure and its relationship to initial infection and inflammation are poorly understood. Universal newborn screening for CF in the United States represents an unprecedented opportunity for investigating CF clinical manifestations very early in life. Recently developed animal models with pulmonary phenotypic manifestations also provide a window into the early consequences of this genetic disorder. For these reasons, the National Heart, Lung, and Blood Institute (NHLBI) convened a working group of extramural experts, entitled "Future Research Directions in Early CF Lung Disease" on September 21-22, 2010, to identify future research directions of great promise in CF. The priority areas identified included (1) exploring pathogenic mechanisms of early CF lung disease; (2) leveraging newborn screening to elucidate the natural history of early lung disease; (3) developing a spectrum of biomarkers of early lung disease that reflects CF pathophysiology, clinical outcome, and response to treatment; (4) exploring the role of genetics/genomics (e.g., modifier genes, gene-environmental interactions, and epigenetics) in early CF pathogenesis; (5) defining early microbiological events in CF lung disease; and (6) elucidating the initial airway inflammatory, remodeling, and repair mechanisms in CF lung disease. [ABSTRACT FROM AUTHOR]
- Published
- 2012
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11. Macromolecular interactions and ion transport in cystic fibrosis.
- Author
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Guggino WB and Banks-Schlegel SP
- Abstract
Cystic fibrosis (CF) is a genetic disease caused by autosomal recessive mutations of the CF transmembrane regulator, CFTR. CFTR functions in the plasma membrane of epithelial cells lining the lung, pancreas, liver, intestines, sweat duct, and the epididymis. The primary problem in CF is that mutations in CFTR affect its ability to be made, processed, and trafficked to the plasma membrane and/or its function as a Cl(-) channel and conductance regulator. Many proteins and processes normally interact with normal CFTR throughout its life cycle and mutant CFTR during the disease process. Understanding the function of these proteins and processes is expected to provide a clearer understanding of how normal CFTR is involved in salt movement and how mutant CFTR is handled by the cell and leads to the pathophysiology of CF. Recently, efforts to find therapies that correct defective CFTR have been intensifying. To facilitate our understanding of normal and mutant CFTR and the identification of new drug targets for developing novel therapies, a panel of experts was convened by the National Heart, Lung, and Blood Institute to explore the critical questions, challenges, and current opportunities to highlight new areas of research that would facilitate a integrated understanding of the processes and proteins that impact CFTR. The meeting highlighted the multiple pathways and interacting proteins involved in CFTR folding and biosynthesis, processing, and trafficking. A number of critical areas for future study were identified. Although these therapies are promising, a big question remains as to whether simply correcting defective CFTR will lead to significant improvement in patient health or whether the symptoms manifested in CF will require therapies in addition to those that target defective CFTR specifically. [ABSTRACT FROM AUTHOR]
- Published
- 2004
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12. A phase I trial of intranasal Moli1901 for cystic fibrosis.
- Author
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Zeitlin PL, Boyle MP, Guggino WB, Molina L, Zeitlin, Pamela L, Boyle, Michael P, Guggino, William B, and Molina, Luis
- Abstract
Background: The peptide drug Moli1901 activates an alternative chloride channel that is present in cystic fibrosis (CF) nasal and airway epithelia. Doing so bypasses the dysfunctional CF transmembrane regulator.Study Objective: To determine whether intranasal Moli1901 is safe, tolerable, and will induce chloride transport in healthy volunteers and CF subjects.Design: A single-blind (to the participant), randomized, placebo-controlled, dose-escalation study of intranasal Moli1901 was performed in four healthy non-CF participants and four participants with CF. Drug or placebo was administered by intranasal superfusion, and nasal potential difference responses were continuously monitored during sequential dose escalations at 1-min intervals from 0.01 through 10 micro mol/L.Results: Neither Moli1901 nor placebo were associated with visible changes such as edema, erythema, drainage, secretions, or ulcer formation. No elevations in lactate dehydrogenase, albumin, or cell counts were observed in nasal lavage fluid after administration. No clinically significant changes in FEV(1) or other toxicity parameters occurred. Changes in the nasal potential difference (NPD) induced by chloride-free, amiloride-containing Ringers solution and by subsequent superfusion with the same solution plus 10 micro mol/L isoproterenol were consistent with both an acute and a sustained change in chloride transport in response to Moli1901. A similar analysis of NPD in the four CF participants demonstrated an acute response that resolved more quickly. A dose-response relationship to Moli1901 was observed in non-CF participants, but a greater range of variability within the CF participants contributed to the lack of a clear dose-response relationship in this group.Conclusion: Moli1901 stimulates chloride transport in normal and CF nasal epithelia in vivo, but may have a shorter duration of action in CF participants. [ABSTRACT FROM AUTHOR]- Published
- 2004
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13. Safety and biological efficacy of an adeno-associated virus vector -- cystic fibrosis transmembrane regulator (AAV-CFTR) in the cystic fibrosis maxillary sinus.
- Author
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Wagner JA, Messner AH, Moran ML, Daifuku R, Kouyama K, Desch JK, Manley S, Norbash AM, Conrad CK, Friborg S, Reynolds T, Guggino WB, Moss RB, Carter BJ, Wine JJ, Flotte TR, and Gardner P
- Published
- 1999
14. Amelioration of airway and GI disease in G551D-CF ferrets by AAV1 and AAV6.
- Author
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Ciobanu C, Yanda M, Zeidan A, Izzi J, Guggino WB, and Cebotaru L
- Subjects
- Animals, Lung metabolism, Lung pathology, Disease Models, Animal, Ferrets, Genetic Therapy methods, Genetic Vectors administration & dosage, Genetic Vectors genetics, Dependovirus genetics, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Cystic Fibrosis therapy, Cystic Fibrosis genetics
- Abstract
Gene therapy for CF has concentrated on targeting the lung. Here we took a different approach by injecting into the cephalic vein and spraying into the trachea of G551D, CF ferrets either AAV1 or 6 containing Δ27-264-CFTR, a truncated version of CFTR. Treatment with the potentiator VX-770 was halted for 7 days before instillation to induce a disease phenotype. Indeed, all ferrets were pancreas-insufficient when they entered the study. Four ferrets (three receiving AAV1 and one AAV6) were necropsied 48 days after vector delivery, and four (three receiving AAV6, one AAV1) were euthanized or died prior to the planned necropsy. AAV1 or AAV6 vector genomes, mRNA expression, and CFTR protein were detected in all tracheal and lung samples and in the liver, pancreas, and ileum of the treated ferrets. Surface and basal airway cells, pancreatic and bile ducts, and ileal crypts and villi were successfully transduced. Obstruction of the airways accompanied by pulmonary hemorrhaging, plugged pancreatic and bile ducts as well as mucous plugs in the ileum were noticed in untreated but absent from transduced ferrets necropsied at 48 days. Transduction of G551D ferrets suggests that a combination of systemic and airway application may be the preferred route of delivery for CF., (© 2024. The Author(s), under exclusive licence to Springer Nature Limited.)
- Published
- 2024
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15. Transduction of Ferret Surface and Basal Cells of Airways, Lung, Liver, and Pancreas via Intratracheal or Intravenous Delivery of Adeno-Associated Virus 1 or 6.
- Author
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Yanda MK, Zeidan A, Ciobanu C, Izzi J, Guggino WB, and Cebotaru L
- Subjects
- Animals, Dependovirus genetics, Lung, Liver, Pancreas, RNA, Messenger, Genetic Vectors genetics, Transduction, Genetic, Ferrets genetics, Cystic Fibrosis
- Abstract
Cystic fibrosis (CF) is potentially treatable by gene therapy. Since the identification of the CF gene, preclinical and clinical trials have concentrated on achieving effective gene therapy targeting the lung. However, the lung has proven to be a formidable barrier to successful gene therapy especially for CF, and many clinical trials failed to achieve efficacy. Recent advances in vector design and adeno-associated virus (AAV) serotypes have increased the chances of success. Given that CF is a multi-organ disease, the goal of this study was to test whether a gene therapy approach involving AAV1 or AAV6 vector delivery via the systemic circulation would at the same time overcome the barrier of lung delivery and transduce organs commonly affected by CF. To accomplish this, we sprayed AAV1 containing green fluorescent protein (GFP) into the trachea or injected it intravenously (IV). We also tested AAV6 injected IV. No adverse events were noted. Ferrets were necropsied 30 days after vector delivery. AAV1 or AAV6 vector genomes, messenger RNA (mRNA) expression, and GFP were detected in all the tracheal and lung samples from the treated animals, whether AAV1 was sprayed into the trachea or injected IV or AAV6 was injected IV. Importantly, both surface epithelial and basal cells of the trachea and lung airways were successfully transduced, regardless of which route of delivery or vector serotype used for transduction. We detected also AAV1 and AAV6 vector genomes, mRNA expression, and GFP in the livers and pancreases, particularly in the acinar cells of the pancreatic duct. These data suggest that gene transfer is attainable in the airways, liver, and pancreas using either serotype, AAV1 or AAV6. Given that these same organs are affected in CF, systemic delivery of AAV may be the preferred route of delivery for a gene therapy for CF.
- Published
- 2023
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16. CFTR and PC2, partners in the primary cilia in autosomal dominant polycystic kidney disease.
- Author
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Yanda MK, Ciobanu C, Guggino WB, and Cebotaru L
- Subjects
- Animals, Mice, Cilia metabolism, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Kidney metabolism, Septins genetics, Septins metabolism, Polycystic Kidney, Autosomal Dominant genetics, Polycystic Kidney, Autosomal Dominant metabolism
- Abstract
Defects in the primary cilium are associated with autosomal dominant polycystic kidney disease (ADPKD). We used a combination of animal models, Western blotting, and confocal microscopy and discovered that CFTR and polycystin 2 (PC2) are both colocalized to the cilium in normal kidneys, with the levels of both being decreased in cystic epithelia. Cilia were longer in CFTR-null mice and in cystic cells in our ADPKD animal models. We examined septin 2, known to play a role in cilia length, to act as a diffusion barrier and to serve as an enhancer of proliferation. We found that septin 2 protein levels were upregulated and colocalized strongly with CFTR in cystic cells. Application of VX-809, the CFTR corrector, restored CFTR and PC2 toward normal in the cilia, decreased the protein levels of septin 2, and drastically reduced septin 2 colocalization with CFTR. Our data suggest that CFTR is present in the cilia and plays a role there, perhaps through its conductance of Cl
- . We also postulate that septin 2 is important for localizing CFTR to the apical membrane in cystic epithelia. NEW & NOTEWORTHY CFTR is present in the primary cilia together with polycystin 2 (PC2). Ablation of CFTR makes cilia longer suggesting that CFTR plays a role there, perhaps through its conductance of Cl.- Published
- 2023
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17. Short-Term Steroid Treatment of Rhesus Macaque Increases Transduction.
- Author
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Yanda MK, Tomar V, Cebotaru CV, Guggino WB, and Cebotaru L
- Subjects
- Animals, Dependovirus metabolism, Genetic Vectors genetics, Green Fluorescent Proteins genetics, Green Fluorescent Proteins metabolism, Macaca mulatta genetics, Macaca mulatta metabolism, Methylprednisolone pharmacology, Methylprednisolone therapeutic use, Steroids, Cystic Fibrosis genetics, Cystic Fibrosis Transmembrane Conductance Regulator genetics
- Abstract
Repeat dosing poses a major hurdle for the development of an adeno-associated virus (AAV)-based gene therapy for cystic fibrosis, in part because of the potential for development of an immune reaction to the AAV1 capsid proteins. Here, to dampen the immune response to AAV1, we treated Rhesus monkeys with methylprednisolone before and after the instillation of two doses of AAV1Δ27-264-CFTR into their airways at 0 and 30 days, followed by a single dose of AAV1-GFP on day 60. Animals were euthanized on day 90, except for one monkey that was sacrificed at 1 year. No adverse events occurred, indicating that the two AAV1 vectors are safe. rAAV1-CFTR and AAV1-GFP vector genomes and mRNA transcripts were detectable in all lung sections and in the liver and pancreas at day 90 and after 1 year at levels comparable with animals necropsied at 90 days. The numbers of vector genomes for cystic fibrosis transmembrane regulator (CFTR) and green fluorescent protein (GFP) detected here were higher than those found in the monkeys infected without methylprednisolone treatment that we tested previously.
1 Also, lung surface and keratin 5-positive basal cells showed higher CFTR and GFP staining than did the cells from the uninfected monkey control. Positive immunostaining, also detected in the liver and pancreas, remained stable for at least a year. All animals seroconverted for anticapsid antibodies by 90 days post-treatment. The neutralizing antibody titer declined in the animal necropsied at 1 year. Conclusion: AAV1 safely and effectively transduces monkey airway and basal cells. Both the presence of vector genomes and transduction from AAV1-CFTR and AAV1-GFP virus seen in the monkeys 4 months to 1 year after the first instillation suggest that repeat dosing with AAV1-based vectors is achievable, particularly after methylprednisolone treatment.- Published
- 2022
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18. The Mitochondrial Ca 2+ import complex is altered in ADPKD.
- Author
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Yanda MK, Tomar V, Cole R, Guggino WB, and Cebotaru L
- Subjects
- Animals, Calcium metabolism, Mice, Mitochondria metabolism, TRPP Cation Channels, Cysts, Polycystic Kidney, Autosomal Dominant genetics
- Abstract
Mutations in either of the polycystic kidney disease genes, PKD1 or PKD2, engender the growth of cysts, altering renal function. Cystic growth is supported by major changes in cellular metabolism, some of which involve the mitochondrion, a major storage site for Ca
2+ and a key organelle in cellular Ca2+ signaling. The goal here was to understand the role of components of the mitochondrial Ca2+ uptake complex in PC1-mutant cells in autosomal dominant polycystic kidney disease (ADPKD). We found that the mitochondrial Ca2+ uniporter (MCU) and voltage-dependent anion channels 1& 3 (VDAC) were down-regulated in different mouse and cell models of ADPKD along with the Ca2+ -dependent enzyme, pyruvate dehydrogenase phosphatase (PDHX). The release of Ca2+ from the endoplasmic reticulum, and Ca2+ uptake by the mitochondria were upregulated in PC1(polycystin)-null cells. We also observed an enhanced staining with MitoTracker Red CMXRos in PC1-null cultured cells than in PC1-containing cells and a substantially higher increase in response to ER Ca2+ release. Increased colocalization of the Ca2+ sensitive dye, rhodamine2, with MitoTracker Green suggested an increase Ca2+ entry into the mitochondria in PC1 null cells subsequent to Ca2+ release from the ER or from Ca2+ entry from the extracellular solution. These data clearly demonstrate abnormal release of Ca2+ by the ER and corresponding alterations in Ca2+ uptake by the mitochondria in PC1- null cells. Importantly, inhibiting mitochondrial Ca2+ uptake with the specific inhibitor Ru360 inhibited cyst growth and altered both apoptosis and cell proliferation. We further show that the decrease in mitochondrial proteins and abnormally high Ca2+ signaling can be reversed by application of the cystic fibrosis (CFTR) corrector, VX-809. We conclude that enhanced Ca2+ signaling and alterations in proteins association with the mitochondrial Ca2+ uptake complex are associated with malfunction of PC1. Finally, our results identify novel therapeutic targets for treating ADPKD., (Copyright © 2021 Elsevier Ltd. All rights reserved.)- Published
- 2022
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19. Megalin-mediated albumin endocytosis in renal proximal tubules is involved in the antiproteinuric effect of angiotensin II type 1 receptor blocker in a subclinical acute kidney injury animal model.
- Author
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Peruchetti DB, Barahuna-Filho PFR, Silva-Aguiar RP, Abreu TP, Takiya CM, Cheng J, Pinheiro AAS, Cebotaru L, Guggino WB, and Caruso-Neves C
- Subjects
- Acute Kidney Injury metabolism, Albumins metabolism, Angiotensin II metabolism, Animals, Cells, Cultured, Disease Models, Animal, Endocytosis drug effects, Kidney Tubules, Proximal metabolism, Low Density Lipoprotein Receptor-Related Protein-2 metabolism, Male, Mice, Mice, Inbred C57BL, Acute Kidney Injury drug therapy, Albumins antagonists & inhibitors, Angiotensin II Type 1 Receptor Blockers pharmacology, Kidney Tubules, Proximal drug effects, Losartan pharmacology, Low Density Lipoprotein Receptor-Related Protein-2 antagonists & inhibitors
- Abstract
Background: Tubule-interstitial injury (TII) is one of the mechanisms involved in the progression of renal diseases with progressive proteinuria. Angiotensin II (Ang II) type 1 receptor blockers (ARBs) have been successfully used to treat renal diseases. However, the mechanism correlating treatment with ARBs and proteinuria is not completely understood. The hypothesis that the anti-proteinuric effect of losartan is associated with the modulation of albumin endocytosis in PT epithelial cells (PTECs) was assessed., Methods: We used a subclinical acute kidney injury animal model (subAKI) and LLC-PK1 cells, a model of PTECs., Results: In subAKI, PT albumin overload induced TII development, measured by: (1) increase in urinary lactate dehydrogenase and γ-glutamyltranspeptidase activity; (2) proteinuria associated with impairment in megalin-mediated albumin reabsorption; (3) increase in luminal and interstitial space in tubular cortical segments. These effects were avoided by treating the animals with losartan, an ARB. Using LLC-PK1 cells, we observed that: (1) 20 mg/mL albumin increased the secretion of Ang II and decreased megalin-mediated albumin endocytosis; (2) the effects of Ang II and albumin were abolished by 10
-8 M losartan; (3) MEK/ERK pathway is the molecular mechanism underlying the Ang II-mediated inhibitory effect of albumin on PT albumin endocytosis., Conclusion: Our results show that PT megalin-mediated albumin endocytosis is a possible target during the treatment of renal diseases patients with ARB., General Significance: The findings obtained in the present work represents a step forward to the current knowledge on about the role of ARBs in the treatment of renal disease., (Copyright © 2021 Elsevier B.V. All rights reserved.)- Published
- 2021
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20. Transduction of Surface and Basal Cells in Rhesus Macaque Lung Following Repeat Dosing with AAV1CFTR.
- Author
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Guggino WB, Yanda MK, Cebotaru CV, and Cebotaru L
- Subjects
- Animals, Cystic Fibrosis genetics, Cystic Fibrosis immunology, Dose-Response Relationship, Drug, Gene Transfer Techniques, Genetic Therapy, Genetic Vectors genetics, Genome, Viral, Humans, Lung immunology, Lung pathology, Macaca mulatta, Cystic Fibrosis therapy, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Dependovirus genetics, Genetic Vectors administration & dosage, Lung metabolism, Transduction, Genetic
- Abstract
To test the effectiveness of repeat dosing, we sprayed two doses (10
13 vg each) of AAV1Δ27-264-CFTR into airways of four rhesus monkeys at 0 and 30 days, followed by a single dose of 1013 vg of AAV1GFP on day 60. Monkeys were sacrificed on day 90. No adverse events occurred, indicating that AAV1 vectors are safe. An elevated anti-AAV1 neutralizing titer was established by the third dose. A positive ELISPOT to the adeno-associated virus (AAV) capsid but not to cystic fibrosis transmembrane conductance regulator (CFTR) occurred after the third dose in three monkeys. AAV1-CFTR and GFP vectors were detectable in all lung sections and in the heart, liver, and spleen. The CFTR protein was higher in treated monkeys than in an untreated monkey. GFP protein was detected in treated lungs. Lung surface and keratin 5-positive basal cells showed higher CFTR staining than in the uninfected monkey and were positive for GFP staining, indicating widespread gene transduction by AAV1CFTR and GFP. AAV1 safely and effectively transduces monkey airway and basal cells. Both the significant numbers of vector genomes and transduction from AAV1CFTR and GFP virus seen in the monkeys 3 months after the first instillation suggest that repeat dosing with AAV1-based vectors is achievable.- Published
- 2020
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21. Gene Therapy for Cystic Fibrosis Paved the Way for the Use of Adeno-Associated Virus in Gene Therapy.
- Author
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Guggino WB and Cebotaru L
- Subjects
- Animals, Gene Transfer Techniques, Genetic Vectors, Humans, Promoter Regions, Genetic, Respiratory System virology, Cystic Fibrosis genetics, Cystic Fibrosis therapy, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Dependovirus genetics, Genetic Therapy
- Abstract
Shortly after the cystic fibrosis (CF) gene was identified in 1989, the race began to develop a gene therapy for this condition. Major efforts utilized full-length cystic fibrosis transmembrane conductance regulator packaged into adenovirus, adeno-associated virus (AAV), or liposomes and delivered to the airways. The drive to find a treatment for CF based on gene therapy drove the early stages of gene therapy in general, particularly those involving AAV gene therapy. Since general overviews of CF gene therapy have already been published, this review considers specifically the efforts using AAV and is focused on honoring the contributions of Dr. Barrie Carter.
- Published
- 2020
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22. A new role for heat shock factor 27 in the pathophysiology of Clostridium difficile toxin B.
- Author
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Yanda MK, Guggino WB, and Cebotaru L
- Subjects
- Caco-2 Cells, Chlorides metabolism, Clostridioides difficile pathogenicity, Clostridium Infections microbiology, Clostridium Infections physiopathology, Colon microbiology, Colon physiopathology, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Diarrhea microbiology, Diarrhea physiopathology, Electric Impedance, Heat-Shock Proteins genetics, Host-Pathogen Interactions, Humans, Molecular Chaperones genetics, Permeability, Protein Binding, Signal Transduction, Tissue Culture Techniques, Bacterial Proteins metabolism, Bacterial Toxins metabolism, Clostridioides difficile metabolism, Clostridium Infections metabolism, Colon innervation, Diarrhea metabolism, Heat-Shock Proteins metabolism, Molecular Chaperones metabolism
- Abstract
Clostridium difficile (CD) is a common pathogen that causes severe gastrointestinal inflammatory diarrhea in patients undergoing antibiotic therapy. Its virulence derives from two toxins, toxin CD, A and B (TcdA and TcdB) (Borriello et al. Rev Infect Dis 12, Suppl 2: S185-191, 1990). Among the prime candidates for CD colonization are patients with cystic fibrosis (CF), who are routinely treated with antibiotics and frequently hospitalized. Indeed, ~50% of patients with CF are colonized with virulent forms of CD but do not exhibit diarrhea (Bauer et al. Clin Microbiol Infect 20: O446-O449, 2014; Binkovitz et al. Am J Roentgenol 172: 517-521, 199; Zemljic et al. Anaerobe 16: 527-532, 2010). We found that TcdB has global effects on colonic cells, including reducing the steady-state levels of sodium-proton exchange regulatory factors, reducing the levels of heat shock protein (Hsp) 27, and increasing the fraction of total Hsp27 bound to the cystic fibrosis transmembrane conductance regulator (CFTR). Also, since some mutations in CFTR seem to be protective, we asked whether CFTR is a target of TcdB. We show here that TcdB increases the maturation of CFTR and transiently increases its function. These combined effects promote increased surface expression of CFTR, resulting in a transient increase in Cl
- secretion. This increase is followed by a precipitous decline in both CFTR-dependent Cl- secretion and transepithelial resistance (TER), suggesting a breakdown in the epithelial cells' tight junctions. We also found that overexpressing Hsp27 reverses some of the deleterious effects of TcdB, in particular preserving TER and therefore likely the maintenance of barrier function. Thus, our data suggest that Hsp27 plays a role in the diarrhea generated by CD infection and is a potential therapeutic target for treating this diarrhea. NEW & NOTEWORTHY Clostridium difficile (CD) is a common pathogen that causes severe gastrointestinal inflammatory diarrhea in patients undergoing antibiotic therapy. We provide new evidence that heat shock protein (Hsp) 27 is one of the key players in CD pathology and that increasing Hsp27 can prevent the decrease in transepithelial resistance induced by toxin CD B, pointing the way for pharmacologic therapies for patients with chronic CD infection that can increase Hsp27 as a means to mitigate the effects of CD on gastrointestinal pathology.- Published
- 2020
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23. Role of calcium in adult onset polycystic kidney disease.
- Author
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Yanda MK, Liu Q, Cebotaru V, Guggino WB, and Cebotaru L
- Subjects
- Animals, Cyclic AMP metabolism, Endoplasmic Reticulum metabolism, Endoplasmic Reticulum pathology, Mice, Inbred C57BL, Polycystic Kidney, Autosomal Dominant pathology, Stromal Interaction Molecule 1 metabolism, TRPP Cation Channels metabolism, Thapsigargin metabolism, Calcium metabolism, Polycystic Kidney, Autosomal Dominant metabolism
- Abstract
Autosomal dominant polycystic kidney disease (ADPKD) is caused by mutations in genes encoding the polycystin (PC) 1 and 2 proteins. The goal of this study was to determine the role of calcium in regulating cyst growth. Stromal interaction molecule 1 (STIM1) protein expression was 15-fold higher in PC1-null proximal tubule cells (PN) than in heterozygote (PH) controls and 2-fold higher in an inducible, PC1 knockout, mouse model of ADPKD compared to a non-cystic match control. IP3 receptor protein expression was also higher in the cystic mice. Knocking down STIM1 with siRNA reduced cyst growth and lowered cAMP levels in PN cells. Fura2 measurements of intracellular Ca
2+ showed higher levels of intracellular Ca2+ , SOCE and thaspigargin-stimulated ER Ca2+ release in PN vs. PH cells. There was a dramatic reduction in thapsigargin-stimulated release of ER Ca2+ following STIM1 silencing or application of 2-APB, consistent with altered ER Ca2+ movement; the protein expression of the Ca2+ -dependent adenylyl cyclases (AC) AC3 and AC6 was up- and down-regulated, respectively. Like STIM1 knockdown, application of the calmodulin inhibitor W7 lowered cAMP levels, further indicating that STIM1 regulates AC3 via Ca2+ We conclude that the high levels of STIM1 in ADPKD cells play a role in supporting cyst growth and promoting high cAMP levels and an increased release of Ca2+ from the ER. Thus, our results provide novel therapeutic targets for treating ADPKD., (Copyright © 2018 Elsevier Inc. All rights reserved.)- Published
- 2019
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24. Restoration of F508-del Function by Transcomplementation: The Partners Meet in the Endoplasmic Reticulum.
- Author
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Bergbower EAS, Sabirzhanova I, Boinot C, Guggino WB, and Cebotaru L
- Subjects
- Cell Line, Cystic Fibrosis therapy, Genetic Therapy, Humans, Respiratory Mucosa cytology, Respiratory Mucosa metabolism, Sequence Deletion, Transfection, Cystic Fibrosis genetics, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Dependovirus genetics, Endoplasmic Reticulum genetics
- Abstract
Background/aims: Because of the small size of adeno-associated virus, AAV, the cystic fibrosis conductance regulator, CFTR, cDNA is too large to fit within AAV and must be truncated. We report here on two truncated versions of CFTR, which, when inserted into AAV1 and used to infect airway cells, rescue F508-del CFTR via transcomplementation. The purpose of this study is to shed light on where in the cell transcomplementation occurs and how it results in close association between the endogenous F508-del and truncated CFTR., Methods: We treated CF airway cells (CFBE41o
- ) with AAV2/1 (AAV2 inverted terminal repeats/AAV1 capsid) containing truncated forms of CFTR, ∆264 and ∆27-264 CFTR, who can restore the function of F508-del by transcomplementation. We addressed the aims of the study using a combination of confocal microscopy and short circuit currents measurements. For the latter, CF bronchial epithelial cells (CFBE) were grown on permeable supports., Results: We show that both F508del and the truncation mutants colocalize in the ER and that both the rescued F508-del and the transcomplementing mutants reach the plasma membrane together. There was significant fluorescence resonance energy transfer (FRET) between F508-del and the transcomplementing mutants within the endoplasmic reticulum (ER), suggesting that transcomplementation occurs through a bimolecular interaction. We found that transcomplementation could increase the Isc in CFBE41o- cells stably expressing additional wt-CFTR or F508-del and in parental CFBE41o- cells expressing endogenous levels of F508-del., Conclusion: We conclude that the functional rescue of F508-del by transcomplementation occurs via a bimolecular interaction that most likely begins in the ER and continues at the plasma membrane. These results come at an opportune time for developing a gene therapy for CF and offer new treatment options for a wide range of CF patients., Competing Interests: None of the authors has any financial interests that pertain directly to this work., (© Copyright by the Author(s). Published by Cell Physiol Biochem Press.)- Published
- 2019
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25. Rescue of CFTR NBD2 mutants N1303K and S1235R is influenced by the functioning of the autophagosome.
- Author
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Liu Q, Sabirzhanova I, Yanda MK, Bergbower EAS, Boinot C, Guggino WB, and Cebotaru L
- Subjects
- Animals, Autophagy, Biological Transport, Blotting, Western, Cell Line, Electric Conductivity, Leucine pharmacology, Mutant Proteins drug effects, Mutant Proteins genetics, Mutation, Small Molecule Libraries pharmacology, Aminopyridines pharmacology, Autophagosomes physiology, Benzodioxoles pharmacology, Cystic Fibrosis drug therapy, Cystic Fibrosis genetics, Cystic Fibrosis Transmembrane Conductance Regulator drug effects, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Leucine analogs & derivatives
- Abstract
The missing phenylalanine at position 508, located in nucleotide-binding domain (NBD1) of the cystic fibrosis transmembrane regulator (CFTR), is the most common cystic fibrosis mutation. Severe disease-causing mutations also occur in NBD2. To provide information on potential therapeutic strategies for mutations in NBD2, we used a combination of biochemical, cell biological and electrophysiological approaches and newly created cell lines to study two disease-causing NBD2 mutants, N1303K and S1235R. We observed that neither was sensitive to E64, a cysteine protease inhibitor. However, further investigation showed that when treated with a combination of correctors, C4 + C18, both mutants also responded to E64. Further exploration to assess aggresome throughput using the autophagy regulator LC3 as a marker showed that, in the absence of correctors, N1303K showed a stalled throughput of LC3-II to the aggresome. The throughput became active again after treatment with the corrector combination C4 + C18. Confocal microscopic studies showed that the N1303K and S1235R mutant proteins both co-localized with LC3, but this co-localization was abolished by the corrector combination and, to a lesser extent, by VX-809. Both the corrector combination and VX-809 increased the CFTR chloride channel function of both mutants. We conclude that correctors have a dual effect, particularly on N1303K: they improve trafficking and function at the plasma membrane and reduce the association with autophagosomes. After treatment with correctors persistent degradation by the autophagosome may limit restoration of function. Thus, mutations in NBD2 of CFTR, in contrast to ΔF508-CFTR, may require additional personalized strategies to rescue them., (Copyright © 2018 European Cystic Fibrosis Society. Published by Elsevier B.V. All rights reserved.)
- Published
- 2018
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26. Wireless control of cellular function by activation of a novel protein responsive to electromagnetic fields.
- Author
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Krishnan V, Park SA, Shin SS, Alon L, Tressler CM, Stokes W, Banerjee J, Sorrell ME, Tian Y, Fridman GY, Celnik P, Pevsner J, Guggino WB, Gilad AA, and Pelled G
- Subjects
- Animals, Calcium metabolism, Cells, Cultured, Fish Proteins genetics, Fish Proteins metabolism, Fishes genetics, HEK293 Cells, Humans, Neurons metabolism, Rats, Rats, Sprague-Dawley, Wireless Technology, Avoidance Learning, Electromagnetic Fields, Fishes physiology
- Abstract
The Kryptopterus bicirrhis (glass catfish) is known to respond to electromagnetic fields (EMF). Here we tested its avoidance behavior in response to static and alternating magnetic fields stimulation. Using expression cloning we identified an electromagnetic perceptive gene (EPG) from the K. bicirrhis encoding a protein that responds to EMF. This EPG gene was cloned and expressed in mammalian cells, neuronal cultures and in rat's brain. Immunohistochemistry showed that the expression of EPG is confined to the mammalian cell membrane. Calcium imaging in mammalian cells and cultured neurons expressing EPG demonstrated that remote activation by EMF significantly increases intracellular calcium concentrations, indicative of cellular excitability. Moreover, wireless magnetic activation of EPG in rat motor cortex induced motor evoked responses of the contralateral forelimb in vivo. Here we report on the development of a new technology for remote, non-invasive modulation of cell function.
- Published
- 2018
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27. Syntaxin 8 and the Endoplasmic Reticulum Processing of ΔF508-CFTR.
- Author
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Sabirzhanova I, Boinot C, Guggino WB, and Cebotaru L
- Subjects
- Cell Line, Cystic Fibrosis genetics, Cystic Fibrosis pathology, Cystic Fibrosis Transmembrane Conductance Regulator analysis, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Endoplasmic Reticulum genetics, Endoplasmic Reticulum pathology, Gene Silencing, Humans, Protein Transport, Proteolysis, Qa-SNARE Proteins analysis, Qa-SNARE Proteins genetics, Cystic Fibrosis metabolism, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Endoplasmic Reticulum metabolism, Qa-SNARE Proteins metabolism
- Abstract
Background/aims: Cystic fibrosis (CF) is a lethal recessive disorder caused by mutations in the CF transmembrane conductance regulator (CFTR). ΔF508, the most common mutation, is a misfolded protein that is retained in the endoplasmic reticulum and degraded, precluding delivery to the cell surface [
1 ]., Methods: Here we use a combination of western blotting, immunoprecipitation, and short circuit current techniques combined with confocal microscopy to address whether the SNARE attachment protein, STX8 plays a role in ΔF508's processing and movement out of the ER., Results: Although the SNARE protein STX8 is thought to be functionally related and primarily localized to early endosomes, we show that silencing of STX8, particularly in the presence of the Vertex corrector molecule C18, rescues ΔF508-CFTR, allowing it to reach the cell surface and increasing CFTR-dependent chloride currents by approximately 2.5-fold over control values. STX8 silencing reduced the binding of quality control protein, Hsp 27, a protein that targets ΔF508-CFTR for sumoylation and subsequent degradation, to ΔF508-CFTR. STX8 silencing increased the levels of Hsp 60 a protein involving in early events in protein folding., Conclusion: STX8 knockdown creates an environment favorable for mature ΔF508 to reach the cell surface. The data also suggest that when present at normal levels, STX8 functions as part of the cell's quality control mechanism., (© 2018 The Author(s). Published by S. Karger AG, Basel.)- Published
- 2018
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28. Histone deacetylase 6 inhibition reduces cysts by decreasing cAMP and Ca 2+ in knock-out mouse models of polycystic kidney disease.
- Author
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Yanda MK, Liu Q, Cebotaru V, Guggino WB, and Cebotaru L
- Subjects
- Animals, Calcium Chelating Agents pharmacology, Calcium Signaling genetics, Cell Line, Cyclic AMP genetics, Cysts genetics, Cysts pathology, Disease Models, Animal, Histone Deacetylase 6, Histone Deacetylases genetics, Mice, Mice, Knockout, Mice, Transgenic, Polycystic Kidney, Autosomal Dominant genetics, Polycystic Kidney, Autosomal Dominant pathology, Protein Kinase C genetics, Protein Kinase C metabolism, TRPP Cation Channels genetics, TRPP Cation Channels metabolism, Thapsigargin pharmacology, Calcium metabolism, Calcium Signaling drug effects, Cyclic AMP metabolism, Cysts enzymology, Histone Deacetylase Inhibitors pharmacokinetics, Histone Deacetylases metabolism, Polycystic Kidney, Autosomal Dominant enzymology
- Abstract
Autosomal dominant polycystic kidney disease (ADPKD) is associated with progressive enlargement of multiple renal cysts, often leading to renal failure that cannot be prevented by a current treatment. Two proteins encoded by two genes are associated with ADPKD: PC1 ( pkd1 ), primarily a signaling molecule, and PC2 ( pkd2 ), a Ca
2+ channel. Dysregulation of cAMP signaling is central to ADPKD, but the molecular mechanism is unresolved. Here, we studied the role of histone deacetylase 6 (HDAC6) in regulating cyst growth to test the possibility that inhibiting HDAC6 might help manage ADPKD. Chemical inhibition of HDAC6 reduced cyst growth in PC1-knock-out mice. In proximal tubule-derived, PC1-knock-out cells, adenylyl cyclase 6 and 3 (AC6 and -3) are both expressed. AC6 protein expression was higher in cells lacking PC1, compared with control cells containing PC1. Intracellular Ca2+ was higher in PC1-knock-out cells than in control cells. HDAC inhibition caused a drop in intracellular Ca2+ and increased ATP-simulated Ca2+ release. HDAC6 inhibition reduced the release of Ca2+ from the endoplasmic reticulum induced by thapsigargin, an inhibitor of endoplasmic reticulum Ca2+ -ATPase. HDAC6 inhibition and treatment of cells with the intracellular Ca2+ chelator 1,2-bis(2-aminophenoxy)ethane- N , N , N ', N '-tetraacetic acid tetrakis(acetoxymethyl ester) reduced cAMP levels in PC1-knock-out cells. Finally, the calmodulin inhibitors W-7 and W-13 reduced cAMP levels, and W-7 reduced cyst growth, suggesting that AC3 is involved in cyst growth regulated by HDAC6. We conclude that HDAC6 inhibition reduces cell growth primarily by reducing intracellular cAMP and Ca2+ levels. Our results provide potential therapeutic targets that may be useful as treatments for ADPKD., (© 2017 by The American Society for Biochemistry and Molecular Biology, Inc.)- Published
- 2017
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29. Adeno-Associated Virus (AAV) gene therapy for cystic fibrosis: current barriers and recent developments.
- Author
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Guggino WB and Cebotaru L
- Subjects
- Animals, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Genetic Vectors genetics, Genetic Vectors metabolism, Humans, Promoter Regions, Genetic, Trans-Splicing, Cystic Fibrosis therapy, Dependovirus genetics, Genetic Therapy
- Abstract
Introduction: Since the cystic fibrosis (CF) gene was discovered in 1989, researchers have worked to develop a gene therapy. One of the most promising and enduring vectors is the AAV, which has been shown to be safe. In particular, several clinical trials have been conducted with AAV serotype 2. All of them detected viral genomes, but identification of mRNA transduction was not consistent; clinical outcomes in Phase II studies were also inconsistent. The lack of a positive outcome has been attributed to a less-than-efficient viral infection by AAV2, a weak transgene promoter and the host immune response to the vector. Areas covered: Herein, the authors focus on AAV gene therapy for CF, evaluating past experience with this approach and identifying ways forward, based on the progress that has already been made in identifying and overcoming the limitations of AAV gene therapy. Expert opinion: Such progress makes it clear that this is an opportune time to push forward toward the development of a gene therapy for CF. Drugs to treat the basic defect in CF represent a remarkable advance but cannot treat a significant cohort of patients with rare mutations. Thus, there is a critical need to develop a gene therapy for those individuals.
- Published
- 2017
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30. A Preclinical Study in Rhesus Macaques for Cystic Fibrosis to Assess Gene Transfer and Transduction by AAV1 and AAV5 with a Dual-Luciferase Reporter System.
- Author
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Guggino WB, Benson J, Seagrave J, Yan Z, Engelhardt J, Gao G, Conlon TJ, and Cebotaru L
- Subjects
- Animals, Female, Gene Transfer Techniques adverse effects, Genes, Reporter, Genetic Therapy adverse effects, Genetic Vectors genetics, Luciferases metabolism, Macaca mulatta, Male, Cystic Fibrosis therapy, Dependovirus genetics, Genetic Therapy methods, Luciferases genetics
- Abstract
Cystic fibrosis (CF) is an autosomal recessive disease that is potentially treatable by gene therapy. Since the identification of the gene encoding CF transmembrane conductance regulator, a number of preclinical and clinical trials have been conducted using the first generation of adeno-associated virus, AAV2. All these studies showed that AAV gene therapy for CF is safe, but clinical benefit was not clearly demonstrated. Thus, a new generation of AAV vectors based on other serotypes is needed to move the field forward. This study tested two AAV serotypes (AAV1 and AAV5) using a dual-luciferase reporter system with firefly and Renilla luciferase genes packaged into AAV1 or AAV5, respectively. Two male and two female Rhesus macaques were each instilled in their lungs with both serotypes using a Penn-Century microsprayer. Both AAV1 and AAV5 vector genomes were detected in all the lung samples when measured at the time of necropsy, 45 days after instillation. However, the vector genome number for AAV1 was at least 10-fold higher than for AAV5. Likewise, luciferase activity was also detected in the same samples at 45 days. AAV1-derived activity was not statistically greater than that derived from AAV5. These data suggest that gene transfer is greater for AAV1 than for AAV5 in macaque lungs. Serum neutralizing antibodies were increased dramatically against both serotypes but were less abundant with AAV1 than with AAV5. No adverse events were noted, again indicating that AAV gene therapy is safe. These results suggest that with more lung-tropic serotypes such as AAV1, new clinical studies of gene therapy using AAV are warranted.
- Published
- 2017
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31. Inhibition of histone deacetylase 6 activity reduces cyst growth in polycystic kidney disease.
- Author
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Cebotaru L, Liu Q, Yanda MK, Boinot C, Outeda P, Huso DL, Watnick T, Guggino WB, and Cebotaru V
- Subjects
- Animals, Cell Proliferation drug effects, Chlorides blood, Chlorides metabolism, Cyclic AMP blood, Disease Models, Animal, Dogs, Down-Regulation, Epithelial Cells metabolism, Female, Histone Deacetylase 6, Histone Deacetylases genetics, Humans, Kidney enzymology, Male, Mice, Mice, Inbred C57BL, Mice, Knockout, Polycystic Kidney, Autosomal Dominant genetics, TRPP Cation Channels genetics, Anilides pharmacology, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Epithelial Cells physiology, Histone Deacetylase Inhibitors pharmacology, Histone Deacetylases metabolism, Hydroxamic Acids pharmacology, Kidney drug effects, Polycystic Kidney, Autosomal Dominant metabolism
- Abstract
Abnormal proliferation of cyst-lining epithelium and increased intracystic fluid secretion via the cystic fibrosis transmembrane conductance regulator (CFTR) are thought to contribute to cyst growth in autosomal dominant polycystic kidney disease (ADPKD). Histone deacetylase 6 (HDAC6) expression and activity are increased in certain cancers, neurodegenerative diseases, and in Pkd1-mutant renal epithelial cells. Inhibition of HDAC6 activity with specific inhibitors slows cancer growth. Here we studied the effect of tubacin, a specific HDAC6 inhibitor, on cyst growth in polycystic kidney disease. Treatment with tubacin prevented cyst formation in MDCK cells, an in vitro model of cystogenesis. Cyclic AMP stimulates cell proliferation and activates intracystic CFTR-mediated chloride secretion in ADPKD. Treatment with tubacin downregulated cyclic AMP levels, inhibited cell proliferation, and inhibited cyclic AMP-activated CFTR chloride currents in MDCK cells. We also found that tubacin reduced cyst growth by inhibiting proliferation of cyst-lining epithelial cells, downregulated cyclic AMP levels, and improved renal function in a Pkd1-conditional mouse model of ADPKD. Thus, HDAC6 could play a role in cyst formation and could serve as a potential therapeutic target in ADPKD., (Copyright © 2016 International Society of Nephrology. Published by Elsevier Inc. All rights reserved.)
- Published
- 2016
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32. Correctors Rescue CFTR Mutations in Nucleotide-Binding Domain 1 (NBD1) by Modulating Proteostasis.
- Author
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Lopes-Pacheco M, Sabirzhanova I, Rapino D, Morales MM, Guggino WB, and Cebotaru L
- Subjects
- Animals, Binding Sites, COS Cells, Chlorocebus aethiops, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, HEK293 Cells, Humans, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Mutation
- Abstract
We evaluated whether small molecule correctors could rescue four nucleotide-binding domain 1 (NBD1) mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene (A455E, S492F, ΔI507, and R560T). We first transfected Cos-7 cells (green monkey kidney cells) with A455E, S492F, ΔI507, or R560T and created HEK-293 (human embryonic kidney cells) cell lines stably expressing these CFTR mutations. The mutants showed lowered protein expression, instability at physiological temperature, and rapid degradation. After treatment with correctors CFFT-002, CFFT-003, C3, C4, and/or C18, the combination of C18+C4 showed the most correction and resulted in increased CFTR residing in the plasma membrane. We found a profound decrease in binding of CFTR to histone deacetylases (HDAC) 6 and 7 and heat shock proteins (Hsps) 27 and 40. Silencing Hsp27 or 40 rescued the mutants, but no additional amount of CFTR was rescued when both proteins were knocked down simultaneously. Thus, CFTR mutations in NBD1 can be rescued by a combination of correctors, and the treatment alters the interaction between mutated CFTR and the endoplasmic reticulum machinery., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)
- Published
- 2016
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33. From CFTR biology toward combinatorial pharmacotherapy: expanded classification of cystic fibrosis mutations.
- Author
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Veit G, Avramescu RG, Chiang AN, Houck SA, Cai Z, Peters KW, Hong JS, Pollard HB, Guggino WB, Balch WE, Skach WR, Cutting GR, Frizzell RA, Sheppard DN, Cyr DM, Sorscher EJ, Brodsky JL, and Lukacs GL
- Subjects
- Animals, Chloride Channel Agonists pharmacology, Chloride Channel Agonists therapeutic use, Cystic Fibrosis classification, Cystic Fibrosis drug therapy, Cystic Fibrosis Transmembrane Conductance Regulator agonists, Genetic Predisposition to Disease, Humans, Ion Channel Gating, Mutation, Missense, Cystic Fibrosis genetics, Cystic Fibrosis Transmembrane Conductance Regulator genetics
- Abstract
More than 2000 mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) have been described that confer a range of molecular cell biological and functional phenotypes. Most of these mutations lead to compromised anion conductance at the apical plasma membrane of secretory epithelia and cause cystic fibrosis (CF) with variable disease severity. Based on the molecular phenotypic complexity of CFTR mutants and their susceptibility to pharmacotherapy, it has been recognized that mutations may impose combinatorial defects in CFTR channel biology. This notion led to the conclusion that the combination of pharmacotherapies addressing single defects (e.g., transcription, translation, folding, and/or gating) may show improved clinical benefit over available low-efficacy monotherapies. Indeed, recent phase 3 clinical trials combining ivacaftor (a gating potentiator) and lumacaftor (a folding corrector) have proven efficacious in CF patients harboring the most common mutation (deletion of residue F508, ΔF508, or Phe508del). This drug combination was recently approved by the U.S. Food and Drug Administration for patients homozygous for ΔF508. Emerging studies of the structural, cell biological, and functional defects caused by rare mutations provide a new framework that reveals a mixture of deficiencies in different CFTR alleles. Establishment of a set of combinatorial categories of the previously defined basic defects in CF alleles will aid the design of even more efficacious therapeutic interventions for CF patients., (© 2016 Veit et al. This article is distributed by The American Society for Cell Biology under license from the author(s). Two months after publication it is available to the public under an Attribution–Noncommercial–Share Alike 3.0 Unported Creative Commons License (http://creativecommons.org/licenses/by-nc-sa/3.0).)
- Published
- 2016
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34. CFTR Controls the Activity of NF-κB by Enhancing the Degradation of TRADD.
- Author
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Wang H, Cebotaru L, Lee HW, Yang Q, Pollard BS, Pollard HB, and Guggino WB
- Subjects
- Adaptor Proteins, Signal Transducing, Carrier Proteins metabolism, Cell Line, Cell Movement drug effects, Golgi Matrix Proteins, HEK293 Cells, Humans, Membrane Proteins metabolism, Membrane Transport Proteins, Protein Binding drug effects, Time Factors, Tumor Necrosis Factor-alpha pharmacology, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, NF-kappa B metabolism, Proteolysis drug effects, TNF Receptor-Associated Death Domain Protein metabolism
- Abstract
Background/aims: Chronic lung infection in cystic fibrosis leads to an inflammatory response that persists because of the chronic presence of bacteria and ultimately leads to a catastrophic failure of lung function., Methods: We use a combination of biochemistry, cell and molecular biology to study the interaction of TRADD, a key adaptor molecule in TNFα signaling, with CFTR in the regulation of NFκB., Results: We show that Wt CFTR binds to and colocalizes with TRADD. TRADD is a key signaling intermediate connecting TNFα with activation of NFκB. By contrast, ΔF508 CFTR does not bind to TRADD. NF-κB activation is higher in CFBE expressing ΔF508 CFTR than in cells expressing Wt CFTR. However, this differential effect is abolished when TRADD levels are knocked down. Transfecting Wt CFTR into CFBE cells reduces NF-κB activity. However the reduction is abolished by the CFTR chloride transport inhibitor-172. Consistently, transfecting in the correctly trafficked CFTR conduction mutants G551D or S341A also fail to reduce NFκB activity. Thus CFTR must be functional if it is to regulate NF-κB activity. We also found that TNFα produced a greater increase in NF-κB activity in CFBE cells than in the same cell when Wt CFTR-corrected. Consistently, the effect is also abolished when TRADD is knocked down by shRNA. Thus, Wt CFTR control of TRADD modulates the physiological activation of NF-κB by TNFα. Based on studies with proteosomal and lysosomal inhibitors, the mechanism by which Wt CFTR, but not ΔF508 CFTR, suppresses TRADD is by lysosomal degradation., Conclusion: We have uncovered a novel mechanism whereby Wt CFTR regulates TNFα signaling by enhancing TRADD degradation. Thus by reducing the levels of TRADD, Wt CFTR suppresses downstream proinflammatory NFκB signaling. By contrast, suppression of NF-κB activation fails in CF cells expressing ΔF508 CFTR., (© 2016 The Author(s) Published by S. Karger AG, Basel.)
- Published
- 2016
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35. Regulation of CFTR Expression and Arginine Vasopressin Activity Are Dependent on Polycystin-1 in Kidney-Derived Cells.
- Author
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de Lemos Barbosa CM, Souza-Menezes J, Amaral AG, Onuchic LF, Cebotaru L, Guggino WB, and Morales MM
- Subjects
- Animals, Antidiuretic Agents pharmacology, Cell Line, Cyclic AMP metabolism, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Epithelial Cells cytology, Epithelial Cells drug effects, Epithelial Cells metabolism, Kidney cytology, Mice, Mice, Inbred CFTR, Polycystic Kidney, Autosomal Dominant genetics, Polycystic Kidney, Autosomal Dominant pathology, RNA, Messenger metabolism, Real-Time Polymerase Chain Reaction, TRPP Cation Channels genetics, Transfection, Arginine Vasopressin pharmacology, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Gene Expression Regulation drug effects, TRPP Cation Channels metabolism
- Abstract
Background: Autosomal dominant polycystic kidney disease (ADPKD) is characterized by the development of multiple, progressive, fluid-filled renal cysts that distort the renal parenchyma, leading to end-stage renal failure, mainly after the fifth decade of life. ADPKD is caused by a mutation in the PKD1 or PKD2 genes that encode polycystin-1 (PC-1) and polycystin-2 (PC-2), respectively. PC-1 is an important regulator of several signaling pathways and PC-2 is a nonselective calcium channel. The CFTR chloride channel is responsible for driving net fluid secretion into the cysts, promoting cyst growth. Arginine vasopressin hormone (AVP), in turn, is capable of increasing cystic intracellular cAMP, contributing to cell proliferation, transepithelial fluid secretion, and therefore to disease progression. The aim of this study was to assess if AVP can modulate CFTR and whether PC-1 plays a role in this potential modulation., Methods: M1 cells, derived from mouse cortical collecting duct, were used in the current work. The cells were treated with 10-7 M AVP hormone and divided into two main groups: transfected cells superexpressing PC-1 (Transf) and cells not transfected (Ctrl). CFTR expression was assessed by immunodetection, CFTR mRNA levels were quantified by quantitative reverse transcription-polymerase chain reaction, and CFTR net ion transport was measured using the Ussing chamber technique., Results: AVP treatment increased the levels of CFTR protein and mRNA. CFTR short-circuit currents were also increased. However, when PC-1 was overexpressed in M1 cells, no increase in any of these parameters was detected., Conclusions: CFTR chloride channel expression is increased by AVP in M1 cells and PC-1 is capable of regulating this modulation., (© 2016 S. Karger AG, Basel.)
- Published
- 2016
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36. STIM1fl/fl Ksp-Cre Mouse has Impaired Renal Water Balance.
- Author
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Cebotaru L, Cebotaru V, Wang H, Arend LJ, and Guggino WB
- Subjects
- Animals, Calcium blood, Calcium urine, Creatinine blood, Creatinine urine, Dietary Proteins administration & dosage, Dietary Proteins pharmacology, Kidney Concentrating Ability genetics, Kinesins genetics, Mice, Knockout, Mice, Transgenic, Renal Insufficiency genetics, Renal Insufficiency metabolism, Stromal Interaction Molecule 1 genetics, Urea blood, Urea urine, Urination drug effects, Water-Electrolyte Balance genetics, Kidney metabolism, Kinesins metabolism, Stromal Interaction Molecule 1 metabolism, Water metabolism
- Abstract
Background/aim: STIM1 is as an essential component in store operated Ca2+ entry. However give the paucity of information on the role of STIM1 in kidney, the aim was to study the function of STIM1 in the medulla of the kidney., Methods: we crossed a Ksp-cre mouse with another mouse containing two loxP sites flanking Exon 6 of STIM1. The Ksp-cre mouse is based upon the Ksp-cadherin gene promoter which expresses cre recombinase in developing nephrons, collecting ducts (SD) and thick ascending limbs (TAL) of the loop of Henle., Results: The offspring of these mice are viable without gross morphological changes, however, we noticed that the STIM1 Ksp-cre knockout mice produced more urine compared to control. To examine this more carefully, we fed mice low (LP) and high protein (HP) diets respectively. When mice were fed HP diet STIM1 ko mice had significantly increased urinary volume and lower specific gravity compared to wt mice. In STIM1 ko mice fed HP diet urine creatinine and urea were significantly lower compared to wt mice fed HP diet, however the fractional excretion was the same., Conclusion: These data support the idea that STIM1 ko mice have impaired urinary concentrating ability when challenged with HP diet is most likely caused by impaired Ca2+-dependent signal transduction through the vasopressin receptor cascade., (© 2016 The Author(s) Published by S. Karger AG, Basel.)
- Published
- 2016
- Full Text
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37. Combination of Correctors Rescue ΔF508-CFTR by Reducing Its Association with Hsp40 and Hsp27.
- Author
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Lopes-Pacheco M, Boinot C, Sabirzhanova I, Morales MM, Guggino WB, and Cebotaru L
- Subjects
- Animals, COS Cells, Chlorocebus aethiops, Cystic Fibrosis Transmembrane Conductance Regulator genetics, HEK293 Cells, Humans, Mutation, Protein Binding, Temperature, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, HSP27 Heat-Shock Proteins metabolism, HSP40 Heat-Shock Proteins metabolism
- Abstract
Correcting the processing of ΔF508-CFTR, the most common mutation in cystic fibrosis, is the major goal in the development of new therapies for this disease. Here, we determined whether ΔF508 could be rescued by a combination of small-molecule correctors, and identified the mechanism by which correctors rescue the trafficking mutant of cystic fibrosis transmembrane conductance regulator (CFTR). We transfected COS-7 cells with ΔF508, created HEK-293 stably expressing ΔF508, and utilized CFBE41o(-) cell lines stably transduced with ΔF508. As shown previously, ΔF508 expressed less protein, was unstable at physiological temperature, and rapidly degraded. When the cells were treated with the combination C18 + C4 the mature C-band was expressed at the cell surface. After treatment with C18 + C4, we saw a lower rate of protein disappearance after translation was stopped with cycloheximide. To understand how this rescue occurs, we evaluated the change in the binding of proteins involved in endoplasmic reticulum-associated degradation, such as Hsp27 (HspB1) and Hsp40 (DnaJ). We saw a dramatic reduction in binding to heat shock proteins 27 and 40 following combined corrector therapy. siRNA experiments confirmed that a reduction in Hsp27 or Hsp40 rescued CFTR in the ΔF508 mutant, but the rescue was not additive or synergistic with C4 + 18 treatment, indicating these correctors shared a common pathway for rescue involving a network of endoplasmic reticulum-associated degradation proteins., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2015
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38. Rescuing Trafficking Mutants of the ATP-binding Cassette Protein, ABCA4, with Small Molecule Correctors as a Treatment for Stargardt Eye Disease.
- Author
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Sabirzhanova I, Lopes Pacheco M, Rapino D, Grover R, Handa JT, Guggino WB, and Cebotaru L
- Subjects
- ATP-Binding Cassette Transporters chemistry, ATP-Binding Cassette Transporters genetics, Amino Acid Sequence, Anilides pharmacology, Cystic Fibrosis Transmembrane Conductance Regulator chemistry, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Enzyme Inhibitors pharmacology, Gene Expression, HEK293 Cells, HSP27 Heat-Shock Proteins genetics, Histone Deacetylase 6, Histone Deacetylases genetics, Humans, Hydroxamic Acids pharmacology, Macrolides pharmacology, Macular Degeneration congenital, Macular Degeneration drug therapy, Macular Degeneration genetics, Macular Degeneration metabolism, Molecular Sequence Data, Mutation, Protein Transport, Proteolysis, Sequence Homology, Amino Acid, Signal Transduction, Stargardt Disease, Transgenes, ATP-Binding Cassette Transporters metabolism, Aminopyridines pharmacology, Benzodioxoles pharmacology, HSP27 Heat-Shock Proteins metabolism, Histone Deacetylases metabolism, Protective Agents pharmacology
- Abstract
Stargardt disease is the most common form of early onset macular degeneration. Mutations in ABCA4, a member of the ATP-binding cassette (ABC) family, are associated with Stargardt disease. Here, we have examined two disease-causing mutations in the NBD1 region of ABCA4, R1108C, and R1129C, which occur within regions of high similarity with CFTR, another ABC transporter gene, which is associated with cystic fibrosis. We show that R1108C and R1129C are both temperature-sensitive processing mutants that engage the cellular quality control mechanism and show a strong interaction with the chaperone Hsp 27. Both mutant proteins also interact with HDCAC6 and are degraded in the aggresome. We also demonstrate that novel corrector compounds that are being tested as treatment for cystic fibrosis, such as VX-809, can rescue the processing of the ABCA4 mutants, particularly their expression at the cell surface, and can reduce their binding to HDAC6. Thus, our data suggest that VX-809 can potentially be developed as a new therapy for Stargardt disease, for which there is currently no treatment., (© 2015 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2015
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39. Rescue of NBD2 mutants N1303K and S1235R of CFTR by small-molecule correctors and transcomplementation.
- Author
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Rapino D, Sabirzhanova I, Lopes-Pacheco M, Grover R, Guggino WB, and Cebotaru L
- Subjects
- Animals, Biological Transport, COS Cells, Chlorocebus aethiops, Cystic Fibrosis pathology, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, HEK293 Cells, Humans, Protein Binding, Cystic Fibrosis drug therapy, Cystic Fibrosis genetics, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Genetic Complementation Test, Mutation genetics, Small Molecule Libraries pharmacology
- Abstract
Although, the most common Cystic Fibrosis mutation, ΔF508, in the cystic fibrosis transmembrane regulator. (CFTR), is located in nucleotide binding domain (NBD1), disease-causing mutations also occur in NBD2. To provide information on potential therapeutic strategies for mutations in NBD2, we studied, using a combination of biochemical approaches and newly created cell lines, two disease-causing NBD2 mutants, N1303K and S1235R. Surprisingly, neither was rescued by low temperature. Inhibition of proteasomes with MG132 or aggresomes with tubacin rescued the immature B and mature C bands of N1303K and S1235R, indicating that degradation occurs via proteasomes and aggresomes. We found no effect of the lysosome inhibitor E64. Thus, our results show that these NBD2 mutants are processing mutants with unique characteristics. Several known correctors developed to rescue ΔF508-CFTR, when applied either alone or in combination, significantly increased the maturation of bands B and C of both NBD 2 mutants. The best correction occurred with the combinations of C4 plus C18 or C3 plus C4. Co-transfection of truncated CFTR (∆27-264) into stably transfected cells was also able to rescue them. This demonstrates for the first time that transcomplementation with a truncated version of CFTR can rescue NBD2 mutants. Our results show that the N1303K mutation has a more profound effect on NBD2 processing than S1235R and that small-molecule correctors increase the maturation of bands B and C in NBD2 mutants. In addition, ∆27-264 was able to transcomplement both NDB2 mutants. We conclude that differences and similarities occur in the impact of mutations on NBD2 when compared to ΔF508-CFTR suggesting that individualized strategies may be needed to restore their function. Finally our results are important because they suggest that gene or corrector molecule therapies either alone or in combination individualized for NBD2 mutants may be beneficial for patients bearing N1303K or S1235R mutations.
- Published
- 2015
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40. Overcoming the cystic fibrosis sputum barrier to leading adeno-associated virus gene therapy vectors.
- Author
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Schuster BS, Kim AJ, Kays JC, Kanzawa MM, Guggino WB, Boyle MP, Rowe SM, Muzyczka N, Suk JS, and Hanes J
- Subjects
- Acetylcysteine pharmacology, Cell Line, Cystic Fibrosis therapy, Dependovirus classification, Dependovirus genetics, Genetic Therapy, HEK293 Cells, Humans, Microscopy, Electron, Scanning, Sputum drug effects, Cystic Fibrosis virology, Dependovirus physiology, Genetic Vectors therapeutic use, Sputum virology
- Abstract
Gene therapy has not yet improved cystic fibrosis (CF) patient lung function in human trials, despite promising preclinical studies. In the human CF lung, inhaled gene vectors must penetrate the viscoelastic secretions coating the airways to reach target cells in the underlying epithelium. We investigated whether CF sputum acts as a barrier to leading adeno-associated virus (AAV) gene vectors, including AAV2, the only serotype tested in CF clinical trials, and AAV1, a leading candidate for future trials. Using multiple particle tracking, we found that sputum strongly impeded diffusion of AAV, regardless of serotype, by adhesive interactions and steric obstruction. Approximately 50% of AAV vectors diffused >1,000-fold more slowly in sputum than in water, with large patient-to-patient variation. We thus tested two strategies to improve AAV diffusion in sputum. We showed that an AAV2 mutant engineered to have reduced heparin binding diffused twice as fast as AAV2 on average, presumably because of reduced adhesion to sputum. We also discovered that the mucolytic N-acetylcysteine could markedly enhance AAV diffusion by altering the sputum microstructure. These studies underscore that sputum is a major barrier to CF gene delivery, and offer strategies for increasing AAV penetration through sputum to improve clinical outcomes.
- Published
- 2014
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41. Mis-regulation of mammalian target of rapamycin (mTOR) complexes induced by albuminuria in proximal tubules.
- Author
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Peruchetti DB, Cheng J, Caruso-Neves C, and Guggino WB
- Subjects
- Albumins pharmacology, Animals, Cell Line, Epithelial Cells drug effects, Epithelial Cells metabolism, Kidney Tubules, Proximal cytology, Low Density Lipoprotein Receptor-Related Protein-2 genetics, Low Density Lipoprotein Receptor-Related Protein-2 metabolism, Mechanistic Target of Rapamycin Complex 1, Mechanistic Target of Rapamycin Complex 2, Mitogen-Activated Protein Kinase 1 metabolism, Mitogen-Activated Protein Kinase 3 metabolism, Multiprotein Complexes genetics, Ribosomal Protein S6 Kinases metabolism, Swine, TOR Serine-Threonine Kinases genetics, Albuminuria metabolism, Kidney Tubules, Proximal metabolism, Multiprotein Complexes metabolism, TOR Serine-Threonine Kinases metabolism
- Abstract
High albumin concentrations in the proximal tubule of the kidney causes tubulointerstitial injury, but how this process occurs is not completely known. To address the signal transduction pathways mis-regulated in renal injury, we studied the modulation of mammalian target of rapamycin (mTOR) complexes by physiologic and pathophysiologic albumin concentrations in proximal tubule cells. Physiologic albumin concentrations activated the PI3K/mTORC2/PKB/mTORC1/S6 kinase (S6K) pathway, but pathophysiologically high albumin concentrations overactivated mTORC1 and inhibited mTORC2 activity. This control process involved the activation of ERK1/2, which promoted the inhibition of TSC2 and activation of S6K. Furthermore, S6K was crucial to promoting the over activation of mTORC1 and inhibition of mTORC2. Megalin expression at the luminal membrane is reduced by high concentrations of albumin. In addition, knockdown of megalin mimicked all the effects of pathophysiologic albumin concentrations, which disrupt normal signal transduction pathways and lead to an overactivation of mTORC1 and inhibition of mTORC2. These data provide new perspectives for understanding the molecular mechanisms behind the effects of albumin on the progression of renal disease., (© 2014 by The American Society for Biochemistry and Molecular Biology, Inc.)
- Published
- 2014
- Full Text
- View/download PDF
42. Lung gene therapy with highly compacted DNA nanoparticles that overcome the mucus barrier.
- Author
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Suk JS, Kim AJ, Trehan K, Schneider CS, Cebotaru L, Woodward OM, Boylan NJ, Boyle MP, Lai SK, Guggino WB, and Hanes J
- Subjects
- Administration, Intranasal, Adult, Animals, CHO Cells, Cricetulus, Cystic Fibrosis metabolism, Cystic Fibrosis therapy, DNA chemistry, Esophagus metabolism, Female, Gastric Mucosa metabolism, Genetic Therapy, Humans, Mice, Inbred BALB C, Nanoparticles chemistry, Polyethylene Glycols chemistry, Polyethyleneimine chemistry, Polylysine chemistry, Young Adult, Cystic Fibrosis Transmembrane Conductance Regulator genetics, DNA administration & dosage, Gene Transfer Techniques, Lung metabolism, Mucus metabolism, Nanoparticles administration & dosage
- Abstract
Inhaled gene carriers must penetrate the highly viscoelastic and adhesive mucus barrier in the airway in order to overcome rapid mucociliary clearance and reach the underlying epithelium; however, even the most widely used viral gene carriers are unable to efficiently do so. We developed two polymeric gene carriers that compact plasmid DNA into small and highly stable nanoparticles with dense polyethylene glycol (PEG) surface coatings. These highly compacted, densely PEG-coated DNA nanoparticles rapidly penetrate human cystic fibrosis (CF) mucus ex vivo and mouse airway mucus ex situ. Intranasal administration of the mucus penetrating DNA nanoparticles greatly enhanced particle distribution, retention and gene transfer in the mouse lung airways compared to conventional gene carriers. Successful delivery of a full-length plasmid encoding the cystic fibrosis transmembrane conductance regulator protein was achieved in the mouse lungs and airway cells, including a primary culture of mucus-covered human airway epithelium grown at air-liquid interface, without causing acute inflammation or toxicity. Highly compacted mucus penetrating DNA nanoparticles hold promise for lung gene therapy., (Copyright © 2014 Elsevier B.V. All rights reserved.)
- Published
- 2014
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43. Polycystin-1 negatively regulates Polycystin-2 expression via the aggresome/autophagosome pathway.
- Author
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Cebotaru V, Cebotaru L, Kim H, Chiaravalli M, Boletta A, Qian F, and Guggino WB
- Subjects
- Animals, Dogs, Down-Regulation, Histone Deacetylase 6, Histone Deacetylases metabolism, Kidney metabolism, Madin Darby Canine Kidney Cells, Metabolic Networks and Pathways, Mice, Polycystic Kidney, Autosomal Dominant genetics, Polycystic Kidney, Autosomal Dominant metabolism, TRPP Cation Channels antagonists & inhibitors, TRPP Cation Channels genetics, Autophagy, Phagosomes, TRPP Cation Channels metabolism
- Abstract
Mutations of the PKD1 and PKD2 genes, encoding polycystin-1 (PC1) and polycystin-2 (PC2), respectively, lead to autosomal dominant polycystic kidney disease. Interestingly, up-regulation or down-regulation of PKD1 or PKD2 leads to polycystic kidney disease in animal models, but their interrelations are not completely understood. We show here that full-length PC1 that interacts with PC2 via a C-terminal coiled-coil domain regulates PC2 expression in vivo and in vitro by down-regulating PC2 expression in a dose-dependent manner. Expression of the pathogenic mutant R4227X, which lacks the C-terminal coiled-coil domain, failed to down-regulate PC2 expression, suggesting that PC1-PC2 interaction is necessary for PC2 regulation. The proteasome and autophagy are two pathways that control protein degradation. Proteins that are not degraded by proteasomes precipitate in the cytoplasm and are transported via histone deacetylase 6 (HDAC6) toward the aggresomes. We found that HDAC6 binds to PC2 and that expression of full-length PC1 accelerates the transport of the HDAC6-PC2 complex toward aggresomes, whereas expression of the R4227X mutant fails to do so. Aggresomes are engulfed by autophagosomes, which then fuse with the lysosome for degradation; this process is also known as autophagy. We have now shown that PC1 overexpression leads to increased degradation of PC2 via autophagy. Interestingly, PC1 does not activate autophagy generally. Thus, we have now uncovered a new pathway suggesting that when PC1 is expressed, PC2 that is not bound to PC1 is directed to aggresomes and subsequently degraded via autophagy, a control mechanism that may play a role in autosomal dominant polycystic kidney disease pathogenesis.
- Published
- 2014
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- View/download PDF
44. Complement yourself: Transcomplementation rescues partially folded mutant proteins.
- Author
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Cebotaru L and Guggino WB
- Abstract
Cystic Fibrosis (CF) is an autosomal disease associated with malfunction in fluid and electrolyte transport across several mucosal membranes. The most common mutation in CF is an in-frame three-base pair deletion that removes a phenylalanine at position 508 in the first nucleotide-binding domain of the cystic fibrosis conductance regulator (CFTR) chloride channel. This mutation has been studied extensively and leads to biosynthetic arrest of the protein in the endoplasmic reticulum and severely reduced channel activity. This review discusses a novel method of rescuing ΔF508 with transcomplementation, which occurs when smaller fragments of CFTR containing the wild-type nucleotide binding domain are co-expressed with the ΔF508 deletion mutant. Transcomplementation rescues the processing and channel activity of ΔF508 and reduces its rate of degradation in airway epithelial cells. To apply transcomplementation as a therapy would require that the cDNA encoding the truncated CFTR be delivered to cells. We also discuss a gene therapeutic approach based on delivery of a truncated form of CFTR to airway cells using adeno-associated viral vectors.
- Published
- 2014
- Full Text
- View/download PDF
45. Correcting the cystic fibrosis disease mutant, A455E CFTR.
- Author
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Cebotaru L, Rapino D, Cebotaru V, and Guggino WB
- Subjects
- Anilides pharmacology, Animals, Boronic Acids pharmacology, Bortezomib, COS Cells, Chlorocebus aethiops, Cycloheximide pharmacology, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Gene Expression, Genetic Complementation Test, Half-Life, Humans, Hydroxamic Acids pharmacology, Leupeptins pharmacology, Plasmids, Proteasome Endopeptidase Complex metabolism, Proteolysis drug effects, Pyrazines pharmacology, Transfection, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Mutation, Proteasome Endopeptidase Complex drug effects, Proteasome Inhibitors pharmacology, Small Molecule Libraries pharmacology
- Abstract
Cystic fibrosis is caused by more than 1000 mutations, the most common being the ΔF508 mutation. These mutations have been divided into five classes [1], with ΔF508 CFTR in class II. Here we have studied the class V mutation A455E. We report that the mature and immature bands of A455E are rapidly degraded primarily by proteasomes; the short protein half-life of this mutant therefore resembles that of ΔF508 CFTR. A455E could be rescued by treatment of the cells with proteasome inhibitors. Furthermore, co-transfection of A455E with the truncation mutant Δ264 CFTR also rescued the mature C band, indicating that A455E can be rescued by transcomplementation. We found that Δ264 CFTR bound to A455E, forming a bimolecular complex. Treatment with the compound correctors C3 and C4 also rescued A455E. These results are significant because they show that although ΔF508 belongs to a different class than A455E, it can be rescued by the same strategies, offering therapeutic promise to patients with Class V mutations.
- Published
- 2014
- Full Text
- View/download PDF
46. Transcomplementation by a truncation mutant of cystic fibrosis transmembrane conductance regulator (CFTR) enhances ΔF508 processing through a biomolecular interaction.
- Author
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Cebotaru L, Woodward O, Cebotaru V, and Guggino WB
- Subjects
- Amino Acid Sequence, Animals, CHO Cells, COS Cells, Chlorocebus aethiops, Cricetinae, Cricetulus, Cystic Fibrosis genetics, Cystic Fibrosis metabolism, Cystic Fibrosis therapy, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Dependovirus, Genetic Complementation Test, Genetic Therapy methods, Genetic Vectors, Humans, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Mutation, Missense, Sequence Deletion
- Abstract
We previously showed that a truncation mutant of CFTR missing the first four transmembrane segments of TMD1, Δ264 CFTR, binds to key elements in the ER quality control mechanism to increase the amounts of the mature C band of both wt and ΔF508 CFTR through transcomplementation. Here, we created a new construct, Δ27-264 CFTR. Even though Δ27-264 CFTR is rapidly degraded in the proteasome, steady state protein can be detected by Western blot. Δ27-264 CFTR can also increase the amounts of the mature C band of both wt and ΔF508 CFTR through transcomplementation. Electrophysiology experiments show that Δ27-264 CFTR can restore chloride channel currents. Further experiments with the conduction mutant S341A show conclusively that currents are indeed generated by rescued channel function of ΔF508 CFTR. Immunoprecipitation studies show that Δ27-264 binds to ΔF508-CFTR, suggesting a bimolecular interaction. Thus the adeno-associated viral vector, rAAV-Δ27-264 CFTR, is a highly promising CF gene therapy vector, because it increases the amount of mature band C protein both from wt and ΔF508 CFTR, and rescues channel activity of ΔF508 CFTR.
- Published
- 2013
- Full Text
- View/download PDF
47. Gout-causing Q141K mutation in ABCG2 leads to instability of the nucleotide-binding domain and can be corrected with small molecules.
- Author
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Woodward OM, Tukaye DN, Cui J, Greenwell P, Constantoulakis LM, Parker BS, Rao A, Köttgen M, Maloney PC, and Guggino WB
- Subjects
- ATP Binding Cassette Transporter, Subfamily G, Member 2, ATP-Binding Cassette Transporters genetics, Amino Acid Substitution, Animals, CHO Cells, Cricetinae, Cricetulus, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Gout drug therapy, Gout genetics, HEK293 Cells, Humans, Hyperuricemia drug therapy, Hyperuricemia genetics, Neoplasm Proteins genetics, Protein Stability, Protein Structure, Secondary, Protein Structure, Tertiary, Xenopus laevis, ATP-Binding Cassette Transporters metabolism, Gout metabolism, Hyperuricemia metabolism, Mutation, Missense, Neoplasm Proteins metabolism
- Abstract
The multidrug ATP-binding cassette, subfamily G, 2 (ABCG2) transporter was recently identified as an important human urate transporter, and a common mutation, a Gln to Lys substitution at position 141 (Q141K), was shown to cause hyperuricemia and gout. The nature of the Q141K defect, however, remains undefined. Here we explore the Q141K ABCG2 mutation using a comparative approach, contrasting it with another disease-causing mutation in an ABC transporter, the deletion of Phe-508 (ΔF508) in the cystic fibrosis transmembrane conductance regulator (CFTR). We found, much like in ΔF508 CFTR, that the Q141K mutation leads to instability in the nucleotide-binding domain (NBD), a defect that translates to significantly decreased protein expression. However, unlike the CFTR mutant, the Q141K mutation does not interfere with the nucleotide-binding domain/intracellular loop interactions. This investigation has also led to the identification of critical residues involved in the protein-protein interactions necessary for the dimerization of ABCG2: Lys-473 (K473) and Phe-142 (F142). Finally, we have demonstrated the utility of using small molecules to correct the Q141K defect in expression and function as a possible therapeutic approach for hyperuricemia and gout.
- Published
- 2013
- Full Text
- View/download PDF
48. The GAP portion of Pseudomonas aeruginosa type III secreted toxin ExoS upregulates total and surface levels of wild type CFTR.
- Author
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Tukaye DN, Kwon SH, and Guggino WB
- Subjects
- ADP Ribose Transferases genetics, Animals, Bacterial Proteins genetics, Bacterial Toxins genetics, Cell Line, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Dogs, Humans, Lysosomes drug effects, Lysosomes metabolism, Macrolides pharmacology, Madin Darby Canine Kidney Cells, Point Mutation, Protein Structure, Tertiary, Sodium-Potassium-Exchanging ATPase metabolism, Transfection, Up-Regulation, ADP Ribose Transferases metabolism, Bacterial Proteins metabolism, Bacterial Toxins metabolism, Pseudomonas aeruginosa metabolism
- Abstract
Background: Pseudomonas aeruginosa (PA) infections account for a large percentage of fatal hospital acquired pneumonias. One of the PA Type III secreted toxin (TTST) ExoS, a bifunctional protein with N-terminal GTPase activating protein (GAP) and C-terminal ADP rybosyl transferase (ADPRT) activities, significantly contributes to PA virulence by targeting small molecular weight G-proteins (SMWGP). In this study, we have looked at one of the mechanisms by which the GAP portion of ExoS (ExoS-GAP) mediates cellular toxicity., Methods: The effects of ExoS-GAP on CFTR trafficking were studied in CFBE41o- Kir 2.2 and MDCK cell lines stably expressing CFTR using a transient transfection system., Results: Transient transfection of ExoS-GAP increased the total and surface protein levels of mature wild type CFTR in epithelial cells stably expressing wild type (WT) CFTR. The effect of ExoS-GAP was specific to CFTR in bronchial epithelial cells since it did not affect the total protein levels of Na(+)/K(+)ATPase, another membrane protein. A point mutation in the ExoS GAP domain (R146K), known to disrupt its catalytic GAP activity, abolished the effect of ExoS-GAP on WT CFTR. Lysosomal inhibition studies with Bafilomycin A1 indicate that ExoS-GAP decreased lysosomal degradation of the mature WT CFTR with concomitant increase in the total levels of mature WT CFTR. However, ExoS-GAP did not increase the total protein levels of ∆F508CFTR., Conclusion: The GAP portion of the PA TTST ExoS increases the total and surface levels of wild type CFTR in vitro mammalian cell system. The effect of ExoS-GAP on WT CFTR total protein levels provides new insight into understanding the virulent pathophysiology of PA infections., (Copyright © 2013 S. Karger AG, Basel.)
- Published
- 2013
- Full Text
- View/download PDF
49. Insulin-like growth factor 1 (IGF-1) enhances the protein expression of CFTR.
- Author
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Lee HW, Cheng J, Kovbasnjuk O, Donowitz M, and Guggino WB
- Subjects
- Blotting, Western, Cell Membrane drug effects, Cell Membrane metabolism, Colforsin pharmacology, Fluorescence Resonance Energy Transfer, Golgi Apparatus drug effects, Golgi Apparatus metabolism, HeLa Cells, Humans, Immunoprecipitation, rho GTP-Binding Proteins metabolism, Cystic Fibrosis Transmembrane Conductance Regulator metabolism, Insulin-Like Growth Factor I pharmacology
- Abstract
Low levels of insulin-like growth factor 1 (IGF-1) have been observed in the serum of cystic fibrosis (CF) patients. However, the effects of low serum IGF-1 on the cystic fibrosis transmembrane conductance regulator (CFTR), whose defective function is the primary cause of cystic fibrosis, have not been studied. Here, we show in human cells that IGF-1 increases the steady-state levels of mature wildtype CFTR in a CFTR-associated ligand (CAL)- and TC10-dependent manner; moreover, IGF-1 increases CFTR-mediated chloride transport. Using an acceptor photobleaching fluorescence resonance energy transfer (FRET) assay, we have confirmed the binding of CAL and CFTR in the Golgi. We also show that CAL overexpression inhibits forskolin-induced increases in the cell-surface expression of CFTR. We found that IGF-1 activates TC10, and active TC10 alters the functional association between CAL and CFTR. Furthermore, IGF-1 and active TC10 can reverse the CAL-mediated reduction in the cell-surface expression of CFTR. IGF-1 does not increase the expression of ΔF508 CFTR, whose processing is arrested in the ER. This finding is consistent with our observation that IGF-1 alters the functional interaction of CAL and CFTR in the Golgi. However, when ΔF508 CFTR is rescued with low temperature or the corrector VRT-325 and proceeds to the Golgi, IGF-1 can increase the expression of the rescued ΔF508 CFTR. Our data support a model indicating that CAL-CFTR binding in the Golgi inhibits CFTR trafficking to the cell surface, leading CFTR to the degradation pathway instead. IGF-1-activated TC10 changes the interaction of CFTR and CAL, allowing CFTR to progress to the plasma membrane. These findings offer a potential strategy using a combinational treatment of IGF-1 and correctors to increase the post-Golgi expression of CFTR in cystic fibrosis patients bearing the ΔF508 mutation.
- Published
- 2013
- Full Text
- View/download PDF
50. Dexamethasone regulates CFTR expression in Calu-3 cells with the involvement of chaperones HSP70 and HSP90.
- Author
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Prota LF, Cebotaru L, Cheng J, Wright J, Vij N, Morales MM, and Guggino WB
- Subjects
- Amino Acid Motifs, Cell Line, Cell Membrane metabolism, Culture Media metabolism, Cycloheximide therapeutic use, Dose-Response Relationship, Drug, Glucocorticoids metabolism, Glucocorticoids pharmacology, Humans, Mifepristone pharmacology, Protein Structure, Tertiary, Real-Time Polymerase Chain Reaction methods, Time Factors, Cystic Fibrosis Transmembrane Conductance Regulator genetics, Dexamethasone pharmacology, HSP70 Heat-Shock Proteins metabolism, HSP90 Heat-Shock Proteins metabolism
- Abstract
Background: Dexamethasone is widely used for pulmonary exacerbation in patients with cystic fibrosis, however, not much is known about the effects of glucocorticoids on the wild-type cystic fibrosis channel transmembrane regulator (CFTR). Our aim was to determine the effects of dexamethasone treatment on wild-type CFTR expression., Methods and Results: Dose-response (1 nM to 10 µM) and time course (3 to 48 h) curves were generated for dexamethasone for mRNA expression in Calu-3 cells using a real-time PCR. Within 24 h, dexamethasone (10 nM) showed a 0.3-fold decrease in CFTR mRNA expression, and a 3.2-fold increase in αENaC mRNA expression compared with control groups. Dexamethasone (10 nM) induced a 1.97-fold increase in the total protein of wild-type CFTR, confirmed by inhibition by mifepristone. To access surface protein expression, biotinylation followed by Western blotting showed that dexamethasone treatment led to a 2.35-fold increase in the amount of CFTR in the cell surface compared with the untreated control groups. Once protein translation was inhibited with cycloheximide, dexamethasone could not increase the amount of CFTR protein. Protein stability was assessed by inhibition of protein synthesis with cycloheximide (50 µg/ml) at different times in cells treated with dexamethasone and in untreated cells. Dexamethasone did not alter the degradation of wild-type CFTR. Assessment of the B band of CFTR within 15 min of metabolic pulse labeling showed a 1.5-fold increase in CFTR protein after treatment with dexamethasone for 24 h. Chaperone 90 (HSP90) binding to CFTR increased 1.55-fold after treatment with dexamethasone for 24 h, whereas chaperone 70 (HSP70) binding decreased 0.30 fold in an immunoprecipitation assay., Conclusion: Mature wild-type CFTR protein is regulated by dexamethasone post transcription, involving cotranslational mechanisms with HSP90 and HSP70, which enhances maturation and expression of wild-type CFTR.
- Published
- 2012
- Full Text
- View/download PDF
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