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WS15.5 Transcomplementation by a truncation mutant of CFTR, Δ27-264 CFTR, rescues both trafficking and chloride channel function of ΔF508 CFTR
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Workshop 15. Sorting out the Salt
WS15.5 Transcomplementation by a truncation mutant of CFTR, D27−264 CFTR, rescues both trafficking and chloride channel function of DF508 CFTR L. Cebotaru1,2 , O. Woodward2 , W. Guggino2 . 1 Johns Hopkins University, Ophtalmology, Baltimore, United States; 2 Johns Hopkins University, Physiology, Baltimore, United States In previous studies we showed that transcomplementation of DF508 by truncated CFTR allows DF508 to progress to the cell surface. Here we study whether D27– 264 CFTR which is missing the first 4 transmembrane segments can also rescue DF508 chloride channel function. Whole cell currents were recorded from CHO cells expressing wt, DF508, D27–264, or both D27–264 and DF508 CFTR. Currents were activated by cpt-cAMP and Forskolin. Importantly no current was generated from unrescued DF508 alone, or from D27–264 alone. Only when both were added did we observe significant whole cell currents which reached to a level near to that of wtCFTR. The currents measured only when both are cotransfected could have originated from either from DF508 or D27–264 CFTR. To determine which one generated the currents, we utilized a CFTR conduction mutant, S341A, which alters the conductance of CFTR. S341A/ wtCFTR showed drastically reduced currents even though the protein was highly expressed. When we studied the double mutant, S341A/DF508 we noted expression of B band but no detectable chloride currents. When we cotransfected S341A /DF508 with D27–264 CFTR we did detect CFTR protein but the choride currents were dramatically reduced showing that the currents observed when DF508 and D27–264 CFTR are cotransfected were generated by rescued DF508. Immunoprecipitation studies showed that D27–264 can bind to DF508 CFTR. Our data show that transcomplementation with D27–264 can rescue both trafficking and chloride channel function of DF508 CFTR most likely via a bimolecular interaction.
Oral Presentations
WS15.6 Upregulation of MicroRNA-148b in cystic fibrosis like lung disease of bENaC-overexpressing mice R. Agrawal1,2,3 , M. Castoldi2,4 , S. Altamura2,4 , M.U. Muckenthaler2,4 , M.A. Mall1,2,3 . 1 University of Heidelberg, Division of Paediatric Pulmonology and Cystic Fibrosis Center, Department of Paediatric III, Heidelberg, Germany; 2 European Molecular Biology Laboratory and University of Heidelberg, Molecular Medicine Partnership Unit, Heidelberg, Germany; 3 Translational Lung Research Center, Department of Translational Pulmonology, Heidelberg, Germany; 4 University of Heidelberg, Department of Paediatric Oncology, Haematology and Immunology, Heidelberg, Germany MicroRNAs (miRNAs) have emerged as a new class of regulators in diverse biological and pathological processes; however, their role in cystic fibrosis (CF) lung disease is poorly understood. To study the potential role of miRNAs in the in vivo pathogenesis of CF-like lung disease we performed miRNA array analysis (miChip) in lung tissue of bENaC-Tg mice and wild type littermate controls. Differentially expressed miRNAs were validated by quantitative real-time PCR and their functional relevance was determined by bioinformatics analysis and in luciferase reporter assays. Tissue specific localization of candidate miRNAs was performed by in situ hybridization. We demonstrate that miR-148b is upregulated in the lungs from bENaC-Tg compared to wild type mice. In situ hybridization showed that miR-148b is predominantly expressed and localized in the conducting airways. Bioinformatics analysis and luciferase assay in Hela cells suggest Mig-6 (mitogen inducible gene-6) as a potential target of miR-148b, a protein previously shown in normal lung development. Our results suggest that miR-148b may be implicated in pathogenesis of CF lung disease and may serve as a novel therapeutic target. Supported by BMBF (DZL).
Workshop 15. Sorting out the Salt
WS15.5 Transcomplementation by a truncation mutant of CFTR, D27−264 CFTR, rescues both trafficking and chloride channel function of DF508 CFTR L. Cebotaru1,2 , O. Woodward2 , W. Guggino2 . 1 Johns Hopkins University, Ophtalmology, Baltimore, United States; 2 Johns Hopkins University, Physiology, Baltimore, United States In previous studies we showed that transcomplementation of DF508 by truncated CFTR allows DF508 to progress to the cell surface. Here we study whether D27– 264 CFTR which is missing the first 4 transmembrane segments can also rescue DF508 chloride channel function. Whole cell currents were recorded from CHO cells expressing wt, DF508, D27–264, or both D27–264 and DF508 CFTR. Currents were activated by cpt-cAMP and Forskolin. Importantly no current was generated from unrescued DF508 alone, or from D27–264 alone. Only when both were added did we observe significant whole cell currents which reached to a level near to that of wtCFTR. The currents measured only when both are cotransfected could have originated from either from DF508 or D27–264 CFTR. To determine which one generated the currents, we utilized a CFTR conduction mutant, S341A, which alters the conductance of CFTR. S341A/ wtCFTR showed drastically reduced currents even though the protein was highly expressed. When we studied the double mutant, S341A/DF508 we noted expression of B band but no detectable chloride currents. When we cotransfected S341A /DF508 with D27–264 CFTR we did detect CFTR protein but the choride currents were dramatically reduced showing that the currents observed when DF508 and D27–264 CFTR are cotransfected were generated by rescued DF508. Immunoprecipitation studies showed that D27–264 can bind to DF508 CFTR. Our data show that transcomplementation with D27–264 can rescue both trafficking and chloride channel function of DF508 CFTR most likely via a bimolecular interaction.
Oral Presentations
WS15.6 Upregulation of MicroRNA-148b in cystic fibrosis like lung disease of bENaC-overexpressing mice R. Agrawal1,2,3 , M. Castoldi2,4 , S. Altamura2,4 , M.U. Muckenthaler2,4 , M.A. Mall1,2,3 . 1 University of Heidelberg, Division of Paediatric Pulmonology and Cystic Fibrosis Center, Department of Paediatric III, Heidelberg, Germany; 2 European Molecular Biology Laboratory and University of Heidelberg, Molecular Medicine Partnership Unit, Heidelberg, Germany; 3 Translational Lung Research Center, Department of Translational Pulmonology, Heidelberg, Germany; 4 University of Heidelberg, Department of Paediatric Oncology, Haematology and Immunology, Heidelberg, Germany MicroRNAs (miRNAs) have emerged as a new class of regulators in diverse biological and pathological processes; however, their role in cystic fibrosis (CF) lung disease is poorly understood. To study the potential role of miRNAs in the in vivo pathogenesis of CF-like lung disease we performed miRNA array analysis (miChip) in lung tissue of bENaC-Tg mice and wild type littermate controls. Differentially expressed miRNAs were validated by quantitative real-time PCR and their functional relevance was determined by bioinformatics analysis and in luciferase reporter assays. Tissue specific localization of candidate miRNAs was performed by in situ hybridization. We demonstrate that miR-148b is upregulated in the lungs from bENaC-Tg compared to wild type mice. In situ hybridization showed that miR-148b is predominantly expressed and localized in the conducting airways. Bioinformatics analysis and luciferase assay in Hela cells suggest Mig-6 (mitogen inducible gene-6) as a potential target of miR-148b, a protein previously shown in normal lung development. Our results suggest that miR-148b may be implicated in pathogenesis of CF lung disease and may serve as a novel therapeutic target. Supported by BMBF (DZL).