(Received for publication, July 13, 1995)
From the
Genetic defects in the cystic fibrosis transmembrane conductance
regulator (CFTR), a cAMP-activated chloride channel, cause cystic
fibrosis. Most defective forms of CFTR show improper intracellular
trafficking. Because isoprenylated, small GTP-binding proteins are
involved in the vesicular trafficking of other integral membrane
proteins, we have investigated the role of isoprenylation in the
trafficking of CFTR to the apical membranes of primary cultures of
human airway epithelium and of Calu-3 cells, a human lung carcinoma
cell line. CFTR function was measured as short circuit current, I efflux, and conductance of cell sheets with
permeabilized basolateral membranes. Lovastatin, an inhibitor of
isoprenyl lipid biosynthesis, markedly inhibited all measures of CFTR
function. The lovastatin-induced declines in CFTR function were
corrected by the simultaneous addition of mevalonate or the isoprenyl
lipids geranylgeranyl and farnesyl but not cholesterol. Lovastatin
reduced total cellular CFTR as assessed by immunoprecipitation.
Mevalonate or isoprenyl lipids protected CFTR levels from the actions
of lovastatin. Together, these results suggest a role for isoprenyl
lipids, presumably through the actions of small GTP-binding proteins,
in the trafficking of CFTR to the apical membrane of human airway
epithelium.
Cystic fibrosis (CF) ()is the commonest lethal
genetic disease in Caucasians. The gene responsible for CF has been
identified (1) and found to encode a 170-kDa glycoprotein known
as the cystic fibrosis transmembrane conductance regulator (CFTR).
Several lines of evidence have suggested that CFTR is a cAMP-regulated
chloride channel in the apical membrane of airway (2, 3) and other epithelial cells(4) . A
relationship between mutations in the CF gene and defective chloride
conductance has been clearly demonstrated(5) .
Although a
variety of mutations in the CFTR gene lead to CF, the commonest
involves a deletion of the phenylalanine residue at the 508 position
(F508)(6) . In transfected cells, many of these CF gene
mutations, including the
F508 variant, result in protein products
that are not completely processed, are retained in the ER, and are
trafficked into a degradation pathway associated with the ER rather
than to the plasma membrane(7, 8) . At reduced
temperatures (<30 °C), CFTR
F508 and other mutant forms are
correctly trafficked to the plasma membrane and confer a cAMP-activated
chloride conductance of similar magnitude to that produced by wild-type
CFTR (9) . Less frequently observed defective forms of CFTR are
trafficked properly to the apical plasma membrane at 37 °C but show
reduced capacity as chloride channels(10) .
Little is known about the trafficking pathways for wild-type CFTR. Previous studies have demonstrated that a class of isoprenylated small GTP-binding proteins (termed Rab proteins) are involved in the trafficking of intracellular vesicles (such as those that would contain CFTR) to the plasma membrane of polarized epithelial cells(11) . A recent report suggests that isoprenylated proteins may be involved in trafficking of CFTR in a colonic carcinoma cell line(12) . The results reported here strongly support a role for isoprenylated proteins in the trafficking of the CFTR to the apical membrane of human airway epithelium.
Additions of lovastatin (Merck Sharp
& Dohme) were made from a 50-fold concentrated ethanolic solution
to produce a final concentration of 50 µM. Mevalonic acid
(Sigma), farnesyl pyrophosphate (F-PP), and geranylgeranyl
pyrophosphate (GG-PP) (American Radiolabeled Chemicals; St. Louis, MO)
were added from stocks prepared in 70% ethanol and 0.075 M
NHHCO
. Cholesterol (Sigma) was added from an
ether stock. The same volumes of vehicles alone were added to control
cells.
Stable base-line
efflux was achieved after an initial 7-min period at room temperature.
Over the next 2 min, perfusion temperature was raised to 37 °C. We
have shown elsewhere (16) that the temperature-induced increase
in I efflux occurs via CFTR. We collected 13 further
effluent samples after the initial seven. All samples were counted on a
counter. The filter and medium remaining in the Swinnex filter
holder at the end of the experiment were pooled and also counted.
Efflux was expressed as the fractional loss of radiolabel over time (i.e. total cell counts lost/min of sampling period divided by
the average total counts present in the cells during that time period).
Lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl
coenzyme A reductase, blocks one of the earliest dedicated steps of the
cholesterol synthetic pathway and inhibits the production of several
metabolically important intermediates (18) (Fig. 1). In
order to determine whether lovastatin inhibited isoprenylation of
proteins in our cells, our first studies investigated the extent to
which post-translational modification of p21 had
occurred. The primary translation product of p21
is
cytosolic. On isoprenylation it becomes membrane-bound and shows
slightly increased mobility on SDS-PAGE(19, 20) . 12 h
after the addition of 50 µM lovastatin to HTE, a
retardation of p21
electrophoretic mobility in SDS-PAGE
was seen in Western blots (Fig. 2), reflecting a reduction in
the levels of membrane-associated Ras and an accumulation of the
cytosolic form as described previously(19) . These results show
that these conditions of lovastatin exposure resulted in a reduction in
the extent of isoprenylation of HTE. Similar results were obtained on
Calu-3 cells (data not shown). Mevalonate is a pathway intermediate
whose production is blocked by lovastatin. When added at 2.5 mM at the same time as lovastatin, conversion of the cytosolic to the
membrane-associated form of p21
was restored (Fig. 2).
Figure 1: Diagram of the dedicated cholesterol synthetic pathway. Note the location of lovastatin inhibition of 3-hydroxy-3-methylglutaryl coenzyme A reductase in the pathway. Also note that the pathway intermediates, mevalonate, F-PP, and GG-PP come after the point of lovastatin inhibition. PP, pyrophospate.
Figure 2:
Lovastatin alters the mobility of
p21 in SDS-PAGE. HTE were exposed for 12 h to
vehicle alone (lane 1), 50 µM lovastatin (lane 2), or 50 µM lovastatin plus 2.5 mM mevalonate (lane 3). Levels of p21
were determined by conventional Western blotting. Locations
of the cytosolic (c-p21
) and
membrane-associated (m-p21
) forms of
p21
are denoted.
Ussing chamber studies provided an initial
assessment of the effects of lovastatin on chloride secretion.
Base-line short circuit current (I) of HTE was
5 µA/cm
but reached 15-20 µA/cm
after stimulation with 10 µM isoproterenol, an
increase reflecting induction of chloride secretion(14) .
Treatment with 50 µM lovastatin completely blocked the
chloride secretion induced by isoproterenol after approximately 12 h of
exposure (Fig. 3). Washout of the lovastatin resulted in
complete recovery of the isoproterenol-induced increase in I
after a further 24 h of incubation (Fig. 3). Lovastatin at all exposure times had no significant
effect on R
(data not shown).
Figure 3:
Lovastatin inhibits isoproterenol-induced
chloride secretion. I responses to 10 µM isoproterenol are shown for HTE treated with 50 µM lovastatin (filled circles), untreated time controls (open circles), and cells recovering from a 24 h exposure to
lovastatin (triangles). The means ± S.E. are shown (n = 4-5).
HTE treated for
12 h with 50 µM lovastatin showed reduced increases in I efflux in response to a temperature change from 22 to
37 °C (Fig. 4A). To evaluate the relative
importance of reduced levels of the 15-carbon farnesyl and 20-carbon
geranylgeranyl isoprenoid intermediates of the cholesterol synthetic
pathways, the pyrophosphate forms of these intermediates (F-PP and
GG-PP) were added to cells at the same time as lovastatin. Comparison
of the increase in
I efflux between the time of
temperature change (8 min) and the maximal increase (11 min)
demonstrated that cells simultaneously exposed to lovastatin plus 2.5
mM mevalonate, 10 µM F-PP, or 10 µM GG-PP were protected from the negative effects of lovastatin (Fig. 4B). Similar results were obtained using Calu-3
cell monolayers (Fig. 5). By contrast, 250 µM cholesterol did not block the inhibition induced by lovastatin
(data not shown).
Figure 4:
Lovastatin inhibits temperature-induced I efflux from HTE. A, effluxes of
I were measured using confluent cell sheets isolated from
the same trachea. Cells were exposed to control buffer (open
circles), 50 µM lovastatin (filled circles),
50 µM lovastatin plus 2.5 mM mevalonate (open
squares), 50 µM lovastatin plus 10 µM GG-PP (filled squares), or 50 µM lovastatin
plus 10 µM F-PP (filled triangles). Temperature
was increased from 22 to 37 °C as indicated by the horizontal
bar. B, increases in efflux between 8 and 11 min. The
means ± S.E. are shown (n = 5). CONT,
control; LOVA, lovastatin; L + M, lovastatin
plus mevalonate; L + G, lovastatin plus GG-PP; L
+ F, lovastatin plus F-PP; *, significantly different from
control; , significantly different from lovastatin
alone.
Figure 5:
Temperature-induced increases in I efflux from Calu-3 cells. Panels and symbols are as for Fig. 4. In B, values are the means ± S.E. (n = 5).
Permeabilizing the basolateral plasma membrane of
airway epithelial cells with nystatin has been used previously as a
means of directly investigating anion transport across the apical
membrane(17) . In the presence of a transepithelial chloride
gradient, I of Calu-3 cells increased on exposure
to nystatin (Fig. 6), reflecting the formation of a chloride
concentration gradient across the apical membrane. Further stimulation
of I
occurred following the addition of
forskolin. This stimulation of I
was inhibited by
the addition of 2 mM diphenyl amino carboxylic acid, a blocker
of the CFTR chloride conductance(4) . Pretreatment of Calu-3
cells with 50 µM lovastatin dramatically inhibited the
increases in I
associated with addition of
nystatin or forskolin (Fig. 6). Calu-3 cells simultaneously
exposed to lovastatin plus 2.5 mM mevalonate were nearly
completely protected from the actions of lovastatin; 10 µM
GG-PP was slightly less protective, and 10 µM F-PP offered
only a moderate amount of protection (Fig. 6).
Figure 6:
Lovastatin inhibits forskolin-induced I responses in nystatin-permeabilized cell
sheets. A, typical I
tracing of
confluent monolayers of Calu-3 cells measured in Ussing chambers. After
a stable base line was achieved, 0.72 mg/ml nystatin was added to the
serosal bath. After another 15 min, 10
M forskolin was added, followed by 2 mM diphenyl amino
carboxylic acid. Top trace, control; bottom trace,
pretreated with lovastatin (50 µM, 12 h). B, mean I
responses of Calu-3 cells following
pre-treatment with no drug (closed circles), 50 µM lovastatin (open circles), or 50 µM lovastatin plus 2.5 mM mevalonate (closed
squares), 10 µM GG-PP (open squares), or 10
µM F-PP (closed triangles). Values represent the
means ± S.E. (n = 5-12). NYS and N, nystatin; F, forskolin; CON, control; D, diphenyl amino carboxylic acid.
Disruption of the cholesterol synthetic pathway by lovastatin has previously been shown to impede intracellular trafficking by removing the farnesyl and geranylgeranyl lipids required for effective membrane association of small GTP-binding proteins(21) . Because treatment with lovastatin resulted in a significant decrease in the cAMP-dependent iodide efflux from Calu-3 cells, it was not surprising that lovastatin (50 µM, 24 h) reduced total CFTR levels of Calu-3 cells to 40 ± 7% of control, as determined by immunoprecipitation (Fig. 7). The observed net reduction of cellular CFTR content under conditions induced by lovastatin suggests that the degradation route(s) for this chloride channel is still competent. The effect of lovastatin on total CFTR content was reversed by 2.5 mM mevalonate (Fig. 7), 10 µM GG-PP, or 10 µM F-PP (data not shown).
Figure 7: Lovastatin treatment reduces total cellular CFTR content. CFTR was measured in Calu-3 cells by immunoprecipitation, phosphorylation, and autoradiography. Lane 1, control. Lysate from untreated cells was put through the experimental protocol, except that no CFTR antibody was added. Lane 2, untreated cells. Lane 3, cells treated with lovastatin (50 µM, 12 h). Lane 4, cells treated with lovastatin and mevalonate (2.5 mM).
Trafficking pathways for CFTR are rapidly being elucidated.
CFTR is synthesized and partially glycosylated in the ER. During
passage through the Golgi, the protein becomes fully glycosylated and
is transported to the apical membrane. However, only 25% of
wild-type CFTR successfully passes from the ER to the Golgi as revealed
by pulse-labeling with [
S]methionine in nonpolar
cells transfected with CFTR cDNA and in epithelial cells naturally
expressing CFTR(24, 25) . The remaining 75% of CFTR
protein is believed to be degraded in the ER(24, 26) .
By contrast, essentially all
F508 CFTR is degraded in the
ER(25, 27, 28, 29) , a defect in
trafficking that is probably due to altered folding of the mutant
protein(27) . Interestingly, the fractions of wild-type and
mutant protein passing from the ER to the Golgi can be altered
pharmacologically. In nonpolar human airway epithelial cell lines,
stimulation of the heteromeric G protein, G
,
reduced the amount of wild-type CFTR trafficking to the apical
membrane(30) . Conversely, inhibition of G
caused the appearance of a cAMP-activated chloride conductance in
the membranes of CF cell lines (30) .
There is also evidence
that CFTR is trafficked to and from the apical membrane by exo- and
endocytosis. First, endosomes contain functional
CFTR(31, 32, 33, 34) . Second,
forskolin-induced increases in I efflux and
transepithelial chloride secretion in T
cells are reduced
by inhibitors of microtubule formation(35) . Third, Prince et al.(36) have shown that CFTR is rapidly
internalized in T
cells or in CFPAC cells transfected with
wild-type CFTR and that the rate of internalization is inhibited by
forskolin. Of interest was the finding that the inhibitory effect of
forskolin on CFTR internalization required passage of chloride through
CFTR; it was not seen in chloride-free (gluconate) medium or in CFPAC
cells transfected with nonfunctional, correctly trafficking mutant CFTR (36) .
It is almost certain that all of these various routes for trafficking of CFTR involve small GTP-binding proteins(21) . Most of these are Rab proteins, which are 21-25 kDa in mass and are cytosolic immediately following their manufacture. However, addition of farnesyl or geranylgeranyl moieties to C-terminal cysteine residues leads to their association with cell membranes(37) . Specifically, the isoprenylated Rab proteins bind GDP and GDP dissociation inhibitor proteins. Binding of GTP displaces GDP and GDP dissociation inhibitor, resulting in the GTP-associated form of the isoprenylated Rab protein, which interacts with a Rab receptor and becomes associated with the membrane(37, 38) . Rab proteins on the surface of vesicles interact with GTPase-activating proteins and specific Rab receptors on the target membrane, leading to membrane fusion. Conversion of GTP to GDP leads to the return of the Rab protein to the cytosol.
A knowledge of which GTP-binding proteins regulate the
various pathways for CFTR movement within airway epithelial cells could
lead to therapies for CF. As an initial approach to this area, we have
determined the changes in CFTR distribution in polarized airway
epithelial cells when the functions of multiple Rab proteins are
disrupted by incubation with lovastatin, which acts to inhibit
synthesis of farnesyl and geranylgeranyl. First, we established that
the dose of lovastatin used (50 µM, 12 h) inhibited
farnesylation of p21. We then showed that this dose
inhibited isoproterenol-induced I
across HTE with
a t
of
5 h. This was not a nonspecific
toxic effect because I
recovered with a t
of
12 h, R
was not
altered, and similar effects were obtained by treating cells with N-acetyl-S-geranylgeranyl-L-cysteine, a
specific blocker of methyl esterification of geranyl geranylated
proteins ( (39) and data not shown).
Transepithelial
chloride secretion (measured as isoproterenol-induced I) reflects the coordinated function of several
transport proteins in both the apical and basolateral membranes (40) , and changes in net chloride secretion need not reflect
changes in CFTR content of the apical membrane. Therefore, we measured
CFTR activity as the temperature-induced increases in
I
efflux. Iodide is not transported by the basolateral NaK
Cl
cotransporter (41, 42) and exits cells only through
chloride channels. Iodide efflux from HTE does not respond to elevation
of cAMP(15, 16) , probably because CFTR is activated
under base-line conditions in these cells. However, the
temperature-dependent increase in
I efflux is inhibited
by diphenyl amino carboxylic acid and blockers of protein kinase A but
not by DIDS or 1,2-bis(2-aminophenoxyl)
ethane-N,N,N`,N`-tetraacetic acid-acetoxymethyl ester. Thus,
iodide flux from these cells is via CFTR rather than calcium-activated
halide channels(16) . When the basolateral membrane of Calu-3
cells is permeabilized with nystatin in the presence of a
transepithelial chloride gradient, then changes in I
and conductance reflect changes in the apical membrane chloride
efflux(17) . In this preparation, there is no evidence for
calcium-activated chloride channels(17) , and therefore
cAMP-induced changes in I
across
nystatin-permeabilized Calu-3 cells provide another assay of CFTR
function.
We found that treatment with lovastatin for 12 h reduced
temperature-induced increases in I efflux to 42 ±
10 and 29 ± 5% of control in Calu-3 cells and HTE, respectively.
When Calu-3 cells were exposed to 50 µM lovastatin for 24
h, the I
of nystatin-permeabilized cells in the
presence of forskolin was 23% of control. Detailed time courses for the
effects of lovastatin on CFTR function were not obtained in these
studies. However, assuming an exponential decline toward zero, these
data suggest that with vesicular trafficking inhibited, the t
for CFTR in the apical membrane is 7-11
h, which is in reasonable agreement with the estimated turnover of
14 h determined for CFTR in T
cells treated with
antisense oligonucleotides to CFTR mRNA(43) . All of the
effects of lovastatin on CFTR function could be wholly or partially
reversed by the addition of mevalonate, GG-PP, or F-PP which allow
cells to regain their protein isoprenylation capabilities. By contrast,
the addition of cholesterol (200 µM, 24 h), which is
downstream of farnesyl and geranylgeranyl in the metabolic pathway (Fig. 1), had no effect on the inhibitory action of lovastatin
(data not shown).
Similar effects of lovastatin on chloride
transport have been reported for T cells(12) . In
these cells lovastatin also abolished I
, though
at longer exposures (3 days) than those used by us. However, unlike our
results, lovastatin markedly decreased R
(to zero
with 10 µM lovastatin for 3 days) in those studies.
Furthermore, recovery from lovastatin exposure was not assessed. When
function of apical membrane cAMP-activated chloride conductance was
assessed in nystatin-permeabilized T
cells, exposure to
2.5 mg/ml for 2 days reduced the cAMP-stimulated change in I
to
50% of control(12) .
Alterations in the cAMP-dependent apical membrane chloride conductance could reflect changes in the properties of individual channels (e.g. unitary conductance or probability of opening) or could be due to changes in channel density. These possibilities can only be definitively distinguished by patch-clamp analysis. However, because lovastatin markedly decreased total CFTR in both Calu-3 and HTE (Fig. 7), we speculate that inhibition of trafficking pathways resulted in decreased CFTR content of the apical membrane.
In summary, a variety of approaches suggest that lovastatin reduces the numbers of cAMP-dependent chloride channels (CFTR) in the apical plasma membrane of human airway cells. These effects could be reversed by mevalonate, farnesyl, or geranylgeranyl (intermediates of the cholesterol synthetic pathway) but not by cholesterol. The most likely explanation for these results is that lovastatin lowers levels of isoprenyl lipids, thereby inhibiting the function of small GTP-binding proteins and disrupting the trafficking of CFTR to the apical membrane. Future studies will be directed at identifying the individual GTP-binding proteins involved in these events.