The University of Chicago1, Neurobiology, Pharmacology, and Physiology, Chicago, IL 60637 CNRS2, Protein Phosphorylation & Human Disease Group, Roscoff, France, 29682
Cystic fibrosis (CF) is the most common fatal genetic disease in the United States. The pathology primarily associated with CF is increased susceptibility to pulmonary infections as well as chronic inflammation of the airways. This is linked to a non-functional chloride channel, CFTR (Cystic Fibrosis Transmembrane Conductance Regulator), and we have shown this channel to be important for the bactericidal activity of alveolar macrophages (AMs). When CFTR is absent or mutated, the ability of AMs to acidify their lysosomal compartment and kill bacteria is significantly decreased. This is likely due to the altered lysosomal chloride conductance which normally acts as a charge-shunt into the lysosome and allows further acidification by the lysosomal VATPase. An acidic environment in the lysosome is necessary for optimal activity of the enzymes responsible for the killing of invading bacteria, and thus CFTR deficiency leads to impaired bactericidal activity.
It has been reported that several cyclin-dependent kinase (CDK) inhibitors including Roscovitine and its analogues restore function to mutant CFTR. We hypothesize that CDK inhibitors rescue lysosomal acidification of AMs isolated from CFTR mutant mice. Roscovitine, currently in Phase II clinical trials as an anticancer drug, has also been shown to have direct effects on voltage gated calcium and potassium channels in addition to being a CDK inhibitor. The efficacy of Roscovitine and its chemical analogs was assessed using ratiometric measurements that employ double conjugates of dextran with two fluorophores, red tetramethylrhodamine (TMR) and green Fluorescein. Dextran traffics to the lysosomal compartment where the intensity of green fluorescence is indicative of pH of the lysosomal lumen while TMR fluorescence is pH-independent and can serve as a measure of the dye load. We performed live cell imaging using a Leica S2 AOBS laser confocal microscope employing a DMIRE2 platform and a 63X (NA 1.4) oil objective on murine AMs. Data were obtained by recording the mean fluorescence of each dye and analyzed using Image J to calculate the TMR/Fluorescein ratio. Lysosomal pH was obtained by interpolation from the calibration curve performed on the same cells using a three-component buffer with ionophores. Lysosomal pH was equalized by incubating cells for 10 min in calibration buffers (ph 4.5-7.5) and 10 μM nigericin, 10 μM valinomycin and 0.1 μM bafilomycin. We have shown that small molecular weight kinase inhibitors such as Roscovitine and its analogues can rescue lysosomal acidification in macrophages with altered or absent CFTR. Incubation of ΔF508 alveolar macrophages with 10 µM R-Roscovitine restored lysosomal pH from 6.06 to wild type levels of 5.35. This mutation, which accounts for 80-95% of CF cases, is characterized by synthesis of functional CFTR, but defective trafficking to the plasma membrane. Identical experiments were also performed on AMs isolated from cftr-/- mice. Here, R-Roscovitine and its enantiomer S-Roscovitine restored lysosomal pH from 6.97 to 5.87 and 6.54, respectively. This demonstrates that Roscovitine rescue of lysosomal acidification is CFTR-independent and that the drug may activate additional compensatory channels to restore CFTR function. These studies suggest the possibility of a therapeutic treatment for cystic fibrosis patients targeting compensatory pathways to correct defective CFTR function.
A. J. Gallan was supported by NIH RO1 GM36823 and a grant from the Cystic Fibrosis Foundation Nelson07G0.
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