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WS4.6 MicroRNA-30s negatively regulate planar cell polarity genes in human cystic fibrosis bronchial epithelial cells
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Workshop 4. New insights in CFTR biology
Oral Presentations
WS4.5 LMTK2 regulates CFTR endocytosis by phosphorylation at CFTR Ser-737
WS4.6 MicroRNA-30s negatively regulate planar cell polarity genes in human cystic fibrosis bronchial epithelial cells
S. Luz1 , K. Cihil2 , P.H. Thibodeau3 , D.L. Brautigan4 , M.D. Amaral1 , C.M. Farinha1 , A. Swiatecka-Urban2 . 1 University of Lisboa, Faculty of Sciences, BioFIG − Center for Biodiversity, Functional and Integrative Genomics, Lisboa, Portugal; 2 Children’s Hospital of Pittsburgh, Department of Nephrology, Pittsburgh, United States; 3 University of Pittsburgh, Department of Cell Biology and Physiology, Pittsburgh, United States; 4 University of Virginia School of Medicine, Center for Cell Signaling and Department of Microbiology, Charlottesville, United States
W. Delbart1 , B. Dhooghe1 , P. Wallemacq1 , P. Lebecque2 , T. Leal1 , S. No¨el1 . 1 Universit´e Catholique de Louvain, Louvain Centre for Toxicology and Applied Pharmacology, Brussels, Belgium; 2 Cliniques Universitaires St Luc, Pediatric
Levels of CFTR at the apical plasma membrane of epithelial cells are regulated by clathrin-mediated endocytosis. However, the protein interactions regulating this process in epithelial cells are poorly understood. Previously, we have shown that SYK and WNK4 kinases play a role in this process regulating CFTR levels at the membrane [1,2]. Lemur Tyrosine Kinase 2 (LMTK2) is a transmembrane protein with serine/threonine kinase activity that has been shown to phosphorylate in vitro the residue Ser-737 of CFTR R domain and suggested to be implicated in the inhibition of CFTR-mediated Cl− transport. Furthermore, LMTK2 binds directly to myosin VI, a motor protein that facilitates CFTR endocytosis. Our aim here was to determine how LMTK2 regulates CFTR endocytosis in polarized human airway epithelial cells (CFBE41o- and Calu-3). Our data show that endogenous LMTK2 co-immunoprecipitates with CFTR. Further, both silencing endogenous LMTK2, or overexpressing a kinase-dead LMTK2 fragment containing the transmembrane and kinase domains, reduce CFTR at plasma membrane levels by attenuating its endocytosis. These results show that LMTK2 facilitates CFTR endocytosis in human airway epithelial cells by a mechanism that may involve phosphorylation of Ser-737. The previously observed role of Ser-737 in inhibiting Cl− currents may thus be due, at least partially, to LMTK2-dependent CFTR endocytosis. Supported by fellowship SFRH/BD/47445/2008 (FCT, Portugal) and grants NIH R01HL090767, R01HL090767−02S1, FCT-PIC/BIA-BCM/112635/2009 and BioFig PEst-OE/BIA/UI4046/2011 centre grant. Reference(s) [1] Luz et al (2011) Mol Cell Biol 31:4392–404. [2] Mendes et al (2011) Mol Cell Biol 31: 4076−86.
Pulmonology & Cystic Fibrosis, Brussels, Belgium Objectives: Mucociliary clearance (MCC), abnormal in CF, is physiologically regulated by epithelial ion transport together with mechanical phenomenon such as cilia movement and cough. Planar Cell Polarity (PCP) is a tightly controlled protein network that drives ciliogenesis and cilia function. Cilia structure and function have been studies in CF. Although the majority of these studies showed no structural abnormality and a normal cilia beat frequency (CBF), it has also been showed that ciliary disorientation, rather than ultrastructural abnormalities or slow CBF, may occur secondary to lung inflammation and result in delayed MCC. We hypothesized that CF HBEs may display abnormalities in PCP network which could further impair coordinated cilia function in the plan of the epithelium. Methods: We profiled PCP genes expression by PCR in HBEs. By quantitative RT-PCR, we determined that expression of CELSR3 and Vangl-1 (Van-Gogh like 1) is down-regulated in CF (F508del/F508del) HBEs as compared to non-CF HBEs. In contrast, Fz3, Fz6, Pk1 and Vangl-2 genes are upregulated in CF cells. In silico analysis revealed that Vangl-1 and Vangl-2, Pk1, Dvl1, Dvl2, and CELSR3 are putative targets of miR-30s family. Expression of miR-30a, miR-30b, miR-30c, miR-30d and miR-30e is elevated is CF-HBEs. Knock-down of miR-30s in CFHBEs significantly increased expression of CELSR3, Fz6, Vangl-1, Pk1, Dvl 1 and Dvl2 but did not modify expression of Fz3, Vangl-2 and Dvl3. Conclusion: Our results validate several PCP genes as miR-30s target genes in HBEs and suggest that decreasing miR-30s expression in CF HBEs could modulate cilia function through restoration of PCP genes expression.
Workshop 4. New insights in CFTR biology
Oral Presentations
WS4.5 LMTK2 regulates CFTR endocytosis by phosphorylation at CFTR Ser-737
WS4.6 MicroRNA-30s negatively regulate planar cell polarity genes in human cystic fibrosis bronchial epithelial cells
S. Luz1 , K. Cihil2 , P.H. Thibodeau3 , D.L. Brautigan4 , M.D. Amaral1 , C.M. Farinha1 , A. Swiatecka-Urban2 . 1 University of Lisboa, Faculty of Sciences, BioFIG − Center for Biodiversity, Functional and Integrative Genomics, Lisboa, Portugal; 2 Children’s Hospital of Pittsburgh, Department of Nephrology, Pittsburgh, United States; 3 University of Pittsburgh, Department of Cell Biology and Physiology, Pittsburgh, United States; 4 University of Virginia School of Medicine, Center for Cell Signaling and Department of Microbiology, Charlottesville, United States
W. Delbart1 , B. Dhooghe1 , P. Wallemacq1 , P. Lebecque2 , T. Leal1 , S. No¨el1 . 1 Universit´e Catholique de Louvain, Louvain Centre for Toxicology and Applied Pharmacology, Brussels, Belgium; 2 Cliniques Universitaires St Luc, Pediatric
Levels of CFTR at the apical plasma membrane of epithelial cells are regulated by clathrin-mediated endocytosis. However, the protein interactions regulating this process in epithelial cells are poorly understood. Previously, we have shown that SYK and WNK4 kinases play a role in this process regulating CFTR levels at the membrane [1,2]. Lemur Tyrosine Kinase 2 (LMTK2) is a transmembrane protein with serine/threonine kinase activity that has been shown to phosphorylate in vitro the residue Ser-737 of CFTR R domain and suggested to be implicated in the inhibition of CFTR-mediated Cl− transport. Furthermore, LMTK2 binds directly to myosin VI, a motor protein that facilitates CFTR endocytosis. Our aim here was to determine how LMTK2 regulates CFTR endocytosis in polarized human airway epithelial cells (CFBE41o- and Calu-3). Our data show that endogenous LMTK2 co-immunoprecipitates with CFTR. Further, both silencing endogenous LMTK2, or overexpressing a kinase-dead LMTK2 fragment containing the transmembrane and kinase domains, reduce CFTR at plasma membrane levels by attenuating its endocytosis. These results show that LMTK2 facilitates CFTR endocytosis in human airway epithelial cells by a mechanism that may involve phosphorylation of Ser-737. The previously observed role of Ser-737 in inhibiting Cl− currents may thus be due, at least partially, to LMTK2-dependent CFTR endocytosis. Supported by fellowship SFRH/BD/47445/2008 (FCT, Portugal) and grants NIH R01HL090767, R01HL090767−02S1, FCT-PIC/BIA-BCM/112635/2009 and BioFig PEst-OE/BIA/UI4046/2011 centre grant. Reference(s) [1] Luz et al (2011) Mol Cell Biol 31:4392–404. [2] Mendes et al (2011) Mol Cell Biol 31: 4076−86.
Pulmonology & Cystic Fibrosis, Brussels, Belgium Objectives: Mucociliary clearance (MCC), abnormal in CF, is physiologically regulated by epithelial ion transport together with mechanical phenomenon such as cilia movement and cough. Planar Cell Polarity (PCP) is a tightly controlled protein network that drives ciliogenesis and cilia function. Cilia structure and function have been studies in CF. Although the majority of these studies showed no structural abnormality and a normal cilia beat frequency (CBF), it has also been showed that ciliary disorientation, rather than ultrastructural abnormalities or slow CBF, may occur secondary to lung inflammation and result in delayed MCC. We hypothesized that CF HBEs may display abnormalities in PCP network which could further impair coordinated cilia function in the plan of the epithelium. Methods: We profiled PCP genes expression by PCR in HBEs. By quantitative RT-PCR, we determined that expression of CELSR3 and Vangl-1 (Van-Gogh like 1) is down-regulated in CF (F508del/F508del) HBEs as compared to non-CF HBEs. In contrast, Fz3, Fz6, Pk1 and Vangl-2 genes are upregulated in CF cells. In silico analysis revealed that Vangl-1 and Vangl-2, Pk1, Dvl1, Dvl2, and CELSR3 are putative targets of miR-30s family. Expression of miR-30a, miR-30b, miR-30c, miR-30d and miR-30e is elevated is CF-HBEs. Knock-down of miR-30s in CFHBEs significantly increased expression of CELSR3, Fz6, Vangl-1, Pk1, Dvl 1 and Dvl2 but did not modify expression of Fz3, Vangl-2 and Dvl3. Conclusion: Our results validate several PCP genes as miR-30s target genes in HBEs and suggest that decreasing miR-30s expression in CF HBEs could modulate cilia function through restoration of PCP genes expression.