Cell Biology

ROSCOVITINE RESCUES IMPAIRED LYSOSOMAL ACIDIFICATION OF ALVEOLAR
MACROPHAGES CAUSED BY ABSENT OR MUTATED CFTR,

[A. J. Gallan^1^]{.underline}, L. V. Deriy^1^, L. Meijer^2^, D. J.
Nelson^1^\*, The University of Chicago^1^, Department of Neurobiology,
Pharmacology, and Physiology, Chicago, IL 60637, CNRS, Protein
Phosphorylation & Human Disease Group, Station Biologique, Roscoff,
France, dnelson@bsd.uchicago.edu

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 V~ATP~ase. 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.