05-583 Sigma-AldrichAnti-CFTR Antibody, clone M3A7

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.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.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.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.

The goal of this study was to determine whether transforming growth factor-β1 (TGF-β1) affects epithelial cells lining the vas deferens, an organ that is universally affected in cystic fibrosis male patients. In PVD9902 cells, which are derived from porcine vas deferens epithelium, TGF-β1 exposure significantly reduced short-circuit current (Isc) stimulated by forskolin or a cell membrane-permeant cAMP analog, 8-pCPT-cAMP, suggesting that TGF-β1 affects targets of the cAMP signaling pathway. Electrophysiological results indicated that TGF-β1 reduces the magnitude of current inhibited by cystic fibrosis transmembrane conductance regulator (CFTR) channel blockers. Real-time RT-PCR revealed that TGF-β1 downregulates the abundance of mRNA coding for CFTR, while biotinylation and Western blot showed that TGF-β1 reduces both total CFTR and apical cell surface CFTR abundance. These results suggest that TGF-β1 causes a reduction in CFTR expression, which limits CFTR-mediated anion secretion. TGF-β1-associated attenuation of anion secretion was abrogated by SB431542, a TGF-β1 receptor I inhibitor. Signaling pathway studies showed that the effect of TGF-β1 on Isc was reduced by SB203580, an inhibitor of p38 mitogen-activated protein kinase (MAPK). TGF-β1 exposure also increased the amount of phospho-p38 MAPK substantially. In addition, anisomycin, a p38 MAPK activator, mimicked the effect of TGF-β1, which further suggests that TGF-β1 affects PVD9902 cells through a p38 MAPK pathway. These observations suggest that TGF-β1, via TGF-β1 receptor I and p38 MAPK signaling, reduces CFTR expression to impair CFTR-mediated anion secretion, which would likely compound the effects associated with mild CFTR mutations and ultimately would compromise male fertility.

Bile formation by the liver is initiated by canalicular transport at the hepatocyte membrane, leading to an increase in ductular bile flow. Thus, bile duct epithelial cells (cholangiocytes), which contribute to the volume and dilution of bile through regulated Cl(-) transport, are exposed to changes in flow and shear force at the apical membrane. The aim of the present study was to determine if fluid flow, or shear stress, is a signal regulating cholangiocyte transport. The results demonstrate that, in human and mouse biliary cells, fluid flow, or shear, increases Cl(-) currents and identify TMEM16A, a Ca(2+)-activated Cl(-) channel, as the operative channel. Furthermore, activation of TMEM16A by flow is dependent on PKCα through a process involving extracellular ATP, binding purinergic P2 receptors, and increases in intracellular Ca(2+) concentration. These studies represent the initial characterization of mechanosensitive Cl(-) currents mediated by TMEM16A. Identification of this novel mechanosensitive secretory pathway provides new insight into bile formation and suggests new therapeutic targets to enhance bile formation in the treatment of cholestatic liver disorders.

Adaptation to hypoxia is an essential physiological response to decrease in tissue oxygenation. This process is primarily under the control of transcriptional activator hypoxia-inducible factor (HIF1). A better understanding of the intracellular HIF1 stabilization pathway would help in management of various diseases characterized by anemia. Among human pathologies, cystic fibrosis disease is characterized by a chronic anemia that is inadequately compensated by the classical erythroid response mediated by the HIF pathway. Because the kidney expresses CFTR and is a master organ involved in the adaptation to hypoxia, we used renal cells to explore the relationship between CFTR and the HIF1-mediated pathway. To monitor the adaptive response to hypoxia, we engineered a hypoxia-induced fluorescent reporter system to determine whether CFTR modulates hypoxia-induced HIF1 stabilization. We show that CFTR is a regulator of HIF stabilization by controlling the intracellular reactive oxygen species (ROS) level through its ability to transport glutathione (a ROS scavenger) out of the cell. Moreover, we demonstrated in a mouse model that both the pharmacological inhibition and the ΔF508 mutation of CFTR lead to an impairment of the adaptive erythroid response to oxygen deprivation. We conclude that CFTR controls HIF stabilization through control of the level of intracellular ROS that act as signaling agents in the HIF-1 pathway.

We have previously shown that despite the presence of mRNA encoding CFTR, renal proximal cells do not exhibit cAMP-sensitive Cl(-) conductance (Rubera I, Tauc M, Bidet M, Poujeol C, Cuiller B, Watrin A, Touret N, Poujeol P. Am J Physiol Renal Physiol 275: F651-F663, 1998). Nevertheless, in these cells, CFTR plays a crucial role in the control of the volume-sensitive outwardly rectifying (VSOR) activated Cl(-) currents during hypotonic shock. The aim of this study was to determine the role of CFTR in the regulation of apoptosis volume decrease (AVD) and the apoptosis phenomenon. For this purpose, renal cells were immortalized from primary cultures of proximal convoluted tubules from cftr(+/+) and cftr(-/-) mice. Apoptosis was induced by staurosporine (STS; 1 microM). Cell volume, Cl(-) conductance, caspase-3 activity, intracellular level of reactive oxygen species (ROS), and glutathione content (GSH/GSSG) were monitored during AVD. In cftr(+/+) cells, AVD and caspase-3 activation were strongly impaired by conventional Cl(-) channel blockers and by a specific CFTR inhibitor (CFTR(inh)-172; 5 microM). STS induced activation of CFTR conductance within 15 min, which was progressively replaced by VSOR Cl(-) currents after 60 min of exposure. In parallel, STS induced an increase in ROS content in the time course of VSOR Cl(-) current activation. This increase was impaired by CFTR(inh)-172 and was not observed in cftr(-/-) cells. Furthermore, the intracellular GSH/GSSG content decreased during STS exposure in cftr(+/+) cells only. In conclusion, CFTR could play a key role in the cascade of events leading to apoptosis. This role probably involves control of the intracellular ROS balance by some CFTR-dependent modulation of GSH concentration.

Defective cAMP-stimulated chloride conductance of the plasma membrane of epithelial cell is the hallmark of cystic fibrosis (CF) and results from mutations in the cystic fibrosis transmembrane conductance regulator, CFTR. In the majority of CF patients, mutations in the CFTR lead to its misfolding and premature degradation at the endoplasmic reticulum (ER). Other mutations impair the cAMP-dependent activation or the ion conductance of CFTR chloride channel. In the present work we identify a novel mechanism leading to reduced expression of CFTR at the cell surface, caused by C-terminal truncations. The phenotype of C-terminally truncated CFTR, representing naturally occurring premature termination and frameshift mutations, were examined in transient and stable heterologous expression systems. Whereas the biosynthesis, processing, and macroscopic chloride channel function of truncated CFTRs are essentially normal, the degradation rate of the mature, complex-glycosylated form is 5- to 6-fold faster than the wild type CFTR. These experiments suggest that the C terminus has a central role in maintaining the metabolic stability of the complex-glycosylated CFTR following its exit from the ER and provide a plausible explanation for the severe phenotype of CF patients harboring C-terminal truncations.