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Effects of Lovastatin on Trafficking of Cystic Fibrosis Transmembrane Conductance Regulator in Human Tracheal Epithelium

Effects of Lovastatin on Trafficking of Cystic Fibrosis Transmembrane Conductance Regulator in... THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 270, No. 42, Issue of October 20, pp. 25102–25106, 1995 © 1995 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Effects of Lovastatin on Trafficking of Cystic Fibrosis Transmembrane Conductance Regulator in Human Tracheal Epithelium* (Received for publication, July 13, 1995) Ben-Quan Shen‡, Jonathan H. Widdicombe‡, and Randall J. Mrsny§ From the ‡Cardiovascular Research Institute, University of California, San Francisco, California 94143-0130, the ‡Children’s Hospital Oakland Research Institute, Oakland, California 94609, and the ‡§Department of Pharmaceutical Research and Development, Genentech, Inc., South San Francisco, California 94080-4980 residue at the 508 position (DF508) (6). In transfected cells, Genetic defects in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chlo- many of these CF gene mutations, including the DF508 variant, ride channel, cause cystic fibrosis. Most defective forms result in protein products that are not completely processed, of CFTR show improper intracellular trafficking. Be- are retained in the ER, and are trafficked into a degradation cause isoprenylated, small GTP-binding proteins are in- pathway associated with the ER rather than to the plasma volved in the vesicular trafficking of other integral membrane (7, 8). At reduced temperatures (,30 °C), CFTR membrane proteins, we have investigated the role of DF508 and other mutant forms are correctly trafficked to the isoprenylation in the trafficking of CFTR to the apical plasma membrane and confer a cAMP-activated chloride con- membranes of primary cultures of human airway epi- ductance of similar magnitude to that produced by wild-type thelium and of Calu-3 cells, a human lung carcinoma cell CFTR (9). Less frequently observed defective forms of CFTR line. CFTR function was measured as short circuit cur- are trafficked properly to the apical plasma membrane at 37 °C rent, I efflux, and conductance of cell sheets with but show reduced capacity as chloride channels (10). permeabilized basolateral membranes. Lovastatin, an Little is known about the trafficking pathways for wild-type inhibitor of isoprenyl lipid biosynthesis, markedly in- CFTR. Previous studies have demonstrated that a class of hibited all measures of CFTR function. The lovastatin- isoprenylated small GTP-binding proteins (termed Rab pro- induced declines in CFTR function were corrected by teins) are involved in the trafficking of intracellular vesicles the simultaneous addition of mevalonate or the isopre- (such as those that would contain CFTR) to the plasma mem- nyl lipids geranylgeranyl and farnesyl but not choles- brane of polarized epithelial cells (11). A recent report suggests terol. Lovastatin reduced total cellular CFTR as that isoprenylated proteins may be involved in trafficking of assessed by immunoprecipitation. Mevalonate or CFTR in a colonic carcinoma cell line (12). The results reported isoprenyl lipids protected CFTR levels from the actions of lovastatin. Together, these results suggest a role for here strongly support a role for isoprenylated proteins in the isoprenyl lipids, presumably through the actions of trafficking of the CFTR to the apical membrane of human small GTP-binding proteins, in the trafficking of CFTR airway epithelium. to the apical membrane of human airway epithelium. MATERIALS AND METHODS Cell Culture—Primary cultures of human tracheal epithelium (HTE) or a human lung carcinoma cell line (Calu-3) were plated at 10 cells/ Cystic fibrosis (CF) is the commonest lethal genetic disease cm and grown to confluency on Transwellt inserts (pore size, 0.45 mm; in Caucasians. The gene responsible for CF has been identified 2 surface area, 1 cm ; Costar, Cambridge, MA) coated with human pla- (1) and found to encode a 170-kDa glycoprotein known as the cental collagen (13). High levels of differentiation of HTE were achieved cystic fibrosis transmembrane conductance regulator (CFTR). by adding media containing 2% Ultraser G to the basolateral side of the insert only, leaving an air-interface at the apical surface (14). HTE Several lines of evidence have suggested that CFTR is a cAMP- cultures were used once the transepithelial resistance (R ) was .250 regulated chloride channel in the apical membrane of airway te ohmszcm , which occurred approximately 5–7 days after plating. Calu-3 (2, 3) and other epithelial cells (4). A relationship between cells were used once a R of .100 ohmszcm was achieved, typically te mutations in the CF gene and defective chloride conductance after 10–14 days of culture. has been clearly demonstrated (5). Additions of lovastatin (Merck Sharp & Dohme) were made from a Although a variety of mutations in the CFTR gene lead to 50-fold concentrated ethanolic solution to produce a final concentration of 50 mM. Mevalonic acid (Sigma), farnesyl pyrophosphate (F-PP), and CF, the commonest involves a deletion of the phenylalanine geranylgeranyl pyrophosphate (GG-PP) (American Radiolabeled Chem- icals; St. Louis, MO) were added from stocks prepared in 70% ethanol * This work was supported by a grant from Cystic Fibrosis Research and 0.075 M NH HCO . Cholesterol (Sigma) was added from an ether 4 3 Inc. (to B.-Q. S.) and by National Institutes of Health Specialized stock. The same volumes of vehicles alone were added to control cells. Center of Research Grant HL-42368. The costs of publication of this Assessment of Ras Isoprenylation—Cells were harvested in Tris- article were defrayed in part by the payment of page charges. This buffered saline (150 mM NaCl and 50 mM Tris-HCl, pH 8.0) containing article must therefore be hereby marked “advertisement” in accordance 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, and 1 mM phen- with 18 U.S.C. Section 1734 solely to indicate this fact. ylmethylsulfonyl fluoride. Lysate (200 mg protein in 100 ml) was mixed To whom correspondence should be addressed: Genentech, Inc., with an equal volume of 2 3 SDS sample buffer (1 3 sample buffer Mailstop #6, 460 Pt. San Bruno Blvd., South San Francisco, CA 94080- consisted of 10% glycerol, 62.5 mM Tris-HCl, pH 6.8, 5% b-mercapto- 4980. Tel.: 415-225-2592; Fax: 415-225-1418. ethanol, 2% SDS, and 0.002% bromphenol blue). Following incubation The abbreviations used are: CF, cystic fibrosis; CFTR, cystic fibrosis at 100 °C for 3 min, protein components were separated by SDS-PAGE transmembrane regulator; DIDS, 4,49-diisothiocyanostilbene-2,29-di- (10–18% gel) and transferred onto a nitrocellulose membrane sulfonic acid; ER, endoplasmic reticulum; F-PP, farnesyl pyrophos- (Schleicher & Schuell) at 4 °C for 60 min in 25 mM Tris-HCl, 150 mM phate; GG-PP, geranylgeranyl pyrophosphate; HTE, human tracheal epithelium; I , short circuit current; PAGE, polyacrylamide gel electro- glycine, 0.05% SDS, and 10% methanol, pH 8.3. After transfer, mem- sc phoresis; R , transepithelial resistance. branes were blocked in 1% nonfat dried milk, 1% bovine serum albu- te This is an Open Access article under the CC BY license. Trafficking of CFTR in Human Tracheal Cells 25103 min, 1% polyvinylpyrolidone 10, and 10 mM Na EDTA in Tris-buffered saline overnight at 4 °C. After two 5-min washes in Tris-buffered saline containing 0.05% Tween 20, the membrane was washed twice in wash buffer (1% nonfat dried milk, 0.5% bovine serum albumin and 0.05% Tween 20 in Tris-buffered saline). The membrane was incubated with 10 mg/ml primary antibody (pan-Ras (Ab-3) mouse monoclonal; Onco- gene Science Inc., Manhasset, NY) in wash buffer at room temperature for 2 h, washed 4 times in wash buffer, and incubated at room temper- ature for 1.5 h in wash buffer containing a goat anti-mouse IgG horse- radish peroxidase-conjugated secondary antibody (Zymed Labs, Inc., South San Francisco, CA) diluted 1:2,000 in wash buffer. After 4 washes with wash buffer, Ras proteins were detected with an enhanced chemi- luminescence kit (Amersham Corp). Measurement of Short Circuit Current (I ) and Transepithelial Re- sc sistance (R )—Cell sheets were mounted in Ussing chambers and te bathed with bicarbonate-buffered Krebs-Henseleit solution (pH 7.4) mixed by a gas lift of 95% O and 5% CO at 37 °C. Transepithelial 2 2 potential differences were clamped to zero, and the resulting I was sc displayed continuously on a pen recorder. Every 20 s the R was te determined from the size of the current deflections resulting from 0.2-s voltage pulses of constant amplitude (0.2–1 mV). Stimulation of cAMP- dependent chloride secretion was achieved by the addition of 10 M FIG.1. Diagram of the dedicated cholesterol synthetic path- isoproterenol or 10 M forskolin. way. Note the location of lovastatin inhibition of 3-hydroxy-3-methyl- Halide Efflux—Effluxes were performed as described (15, 16). Cell glutaryl coenzyme A reductase in the pathway. Also note that the sheets were loaded with I by placing them in serum-free medium 125 pathway intermediates, mevalonate, F-PP, and GG-PP come after the containing Na I (10 mCi/ml, 3 nM, and 17.4 Ci/mg; DuPont NEN) for point of lovastatin inhibition. PP, pyrophospate. 2 h. Residual surface-associated tracer was removed with two 200-ml washes (15 s each) using Krebs-Henseleit solution, and monolayers were placed, mucosal side up, in the top half of a Swinnex 25-mm filter holder (Millipore Corp. Bedford, MA) containing a 0.65-mm pore, type D, cellulose ester filter. Oxygenated Krebs-Henseleit solution was passed over the mucosal surface of the monolayers and through surrounding pores in the filter holder at 1 ml/min using a peristaltic pump. Effluent fractions were collected at 1-min intervals. The perfusate temperature was raised to 37 °C using a heated copper wire wrapped around the glass inlet tubing. This wire connected to a temperature controller, which responded to a thermocouple inside the Swinnex filter holder. ras FIG.2. Lovastatin alters the mobility of p21 in SDS-PAGE. Stable base-line efflux was achieved after an initial 7-min period at HTE were exposed for 12 h to vehicle alone (lane 1), 50 mM lovastatin room temperature. Over the next 2 min, perfusion temperature was (lane 2), or 50 mM lovastatin plus 2.5 mM mevalonate (lane 3). Levels of raised to 37 °C. We have shown elsewhere (16) that the temperature- ras p21 were determined by conventional Western blotting. Locations of induced increase in I efflux occurs via CFTR. We collected 13 further ras ras the cytosolic (c-p21 ) and membrane-associated (m-p21 ) forms of effluent samples after the initial seven. All samples were counted on a ras p21 are denoted. g 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 ras the addition of 50 mM lovastatin to HTE, a retardation of p21 counts lost/min of sampling period divided by the average total counts electrophoretic mobility in SDS-PAGE was seen in Western present in the cells during that time period). blots (Fig. 2), reflecting a reduction in the levels of membrane- Nystatin Permeabilization—Cell sheets were bathed with a Krebs- associated Ras and an accumulation of the cytosolic form as Henseleit solution on their serosal side (120 mM NaCl) and on their mucosal sides with a solution in which all but 20 mM NaCl was replaced described previously (19). These results show that these condi- by sodium gluconate. Nystatin (0.72 mg/ml; Sigma) was added to the tions of lovastatin exposure resulted in a reduction in the serosal bath to permeabilize the basolateral membranes. Under these extent of isoprenylation of HTE. Similar results were obtained circumstances, changes in R and I reflect apical membrane chloride te sc on Calu-3 cells (data not shown). Mevalonate is a pathway channel activity (17). intermediate whose production is blocked by lovastatin. When Immunoprecipitation of CFTR—Our methods are described in full added at 2.5 mM at the same time as lovastatin, conversion of elsewhere (13). In brief, cell sheets were lysed with 0.1% SDS, Triton ras X-100, and sodium deoxycholate; samples of lysate were incubated with the cytosolic to the membrane-associated form of p21 was a mouse monoclonal antibody raised against a C terminus peptide of restored (Fig. 2). human CFTR (Genzyme, Cambridge, MA). Antibody-antigen complexes Ussing chamber studies provided an initial assessment of were then adsorbed onto pansorbin cells (Calbiochem) and pelleted. the effects of lovastatin on chloride secretion. Base-line short CFTR was then phosphorylated by adding protein kinase A catalytic circuit current (I ) of HTE was ;5 mA/cm but reached 15–20 subunit (Sigma) and [g- P]ATP (30 Ci/mmol; 2 mCi/ml; DuPont NEN). sc mA/cm after stimulation with 10 mM isoproterenol, an increase Phosphoproteins were separated by 6% SDS-PAGE, and levels of P were determined by densitometric scans of autoradiographs. reflecting induction of chloride secretion (14). Treatment with 50 mM lovastatin completely blocked the chloride secretion in- RESULTS duced by isoproterenol after approximately 12 h of exposure Lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coen- (Fig. 3). Washout of the lovastatin resulted in complete recov- zyme A reductase, blocks one of the earliest dedicated steps of ery of the isoproterenol-induced increase in I after a further sc the cholesterol synthetic pathway and inhibits the production 24 h of incubation (Fig. 3). Lovastatin at all exposure times had of several metabolically important intermediates (18) (Fig. 1). no significant effect on R (data not shown). te In order to determine whether lovastatin inhibited isoprenyla- HTE treated for 12 h with 50 mM lovastatin showed reduced tion of proteins in our cells, our first studies investigated the increases in I efflux in response to a temperature change ras extent to which post-translational modification of p21 had from 22 to 37 °C (Fig. 4A). To evaluate the relative importance ras occurred. The primary translation product of p21 is cytosolic. of reduced levels of the 15-carbon farnesyl and 20-carbon gera- On isoprenylation it becomes membrane-bound and shows nylgeranyl isoprenoid intermediates of the cholesterol syn- slightly increased mobility on SDS-PAGE (19, 20). 12 h after thetic pathways, the pyrophosphate forms of these intermedi- 25104 Trafficking of CFTR in Human Tracheal Cells FIG.3. Lovastatin inhibits isoproterenol-induced chloride se- cretion. I responses to 10 mM isoproterenol are shown for HTE treated sc with 50 mM lovastatin (filled circles), untreated time controls (open circles), and cells recovering from a 24 h exposure to lovastatin (trian- gles). The means 6 S.E. are shown (n 5 4–5). FIG.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 6 S.E. (n 5 5). block the inhibition induced by lovastatin (data not shown). 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 ap- ical membrane (17). In the presence of a transepithelial chlo- ride gradient, I of Calu-3 cells increased on exposure to nys- sc tatin (Fig. 6), reflecting the formation of a chloride concentration gradient across the apical membrane. Further stimulation of I occurred following the addition of forskolin. sc This stimulation of I was inhibited by the addition of 2 mM sc diphenyl amino carboxylic acid, a blocker of the CFTR chloride conductance (4). Pretreatment of Calu-3 cells with 50 mM lov- astatin dramatically inhibited the increases in I associated sc 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 mM GG-PP was slightly less protective, and 10 mM F-PP offered only a moderate amount of protection (Fig. 6). Disruption of the cholesterol synthetic pathway by lovastatin has previously been shown to impede intracellular trafficking by removing the farnesyl and geranylgeranyl lipids required FIG.4. Lovastatin inhibits temperature-induced I efflux for effective membrane association of small GTP-binding pro- from HTE. A, effluxes of I were measured using confluent cell sheets teins (21). Because treatment with lovastatin resulted in a isolated from the same trachea. Cells were exposed to control buffer significant decrease in the cAMP-dependent iodide efflux from (open circles), 50 mM lovastatin (filled circles), 50 mM lovastatin plus 2.5 Calu-3 cells, it was not surprising that lovastatin (50 mM,24h) mM mevalonate (open squares), 50 mM lovastatin plus 10 mM GG-PP (filled squares), or 50 mM lovastatin plus 10 mM F-PP (filled triangles). reduced total CFTR levels of Calu-3 cells to 40 6 7% of control, Temperature was increased from 22 to 37 °C as indicated by the hori- as determined by immunoprecipitation (Fig. 7). The observed zontal bar. B, increases in efflux between 8 and 11 min. The means 6 net reduction of cellular CFTR content under conditions in- S.E. are shown (n 5 5). CONT, control; LOVA, lovastatin; L 1 M, duced by lovastatin suggests that the degradation route(s) for lovastatin plus mevalonate; L 1 G, lovastatin plus GG-PP; L 1 F, lovastatin plus F-PP; *, significantly different from control; †, signifi- this chloride channel is still competent. The effect of lovastatin cantly different from lovastatin alone. on total CFTR content was reversed by 2.5 mM mevalonate (Fig. 7), 10 mM GG-PP, or 10 mM F-PP (data not shown). ates (F-PP and GG-PP) were added to cells at the same time as 125 DISCUSSION lovastatin. Comparison of the increase in I efflux between the time of temperature change (8 min) and the maximal in- Trafficking pathways for CFTR are rapidly being elucidated. crease (11 min) demonstrated that cells simultaneously ex- CFTR is synthesized and partially glycosylated in the ER. posed to lovastatin plus 2.5 mM mevalonate, 10 mM F-PP, or 10 During passage through the Golgi, the protein becomes fully mM GG-PP were protected from the negative effects of lovasta- glycosylated and is transported to the apical membrane. How- tin (Fig. 4B). Similar results were obtained using Calu-3 cell ever, only ;25% of wild-type CFTR successfully passes from monolayers (Fig. 5). By contrast, 250 mM cholesterol did not the ER to the Golgi as revealed by pulse-labeling with [ S]me- Trafficking of CFTR in Human Tracheal Cells 25105 FIG.7. Lovastatin treatment reduces total cellular CFTR con- tent. 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 mM,12h). Lane 4, cells treated with lovas- tatin and mevalonate (2.5 mM). 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 re- ceptors on the target membrane, leading to membrane fusion. Conversion of GTP to GDP leads to the return of the Rab FIG.6. Lovastatin inhibits forskolin-induced I responses in sc nystatin-permeabilized cell sheets. A, typical I tracing of conflu- protein to the cytosol. sc ent monolayers of Calu-3 cells measured in Ussing chambers. After a A knowledge of which GTP-binding proteins regulate the stable base line was achieved, 0.72 mg/ml nystatin was added to the various pathways for CFTR movement within airway epithelial serosal bath. After another 15 min, 10 M forskolin was added, fol- cells could lead to therapies for CF. As an initial approach to lowed by 2 mM diphenyl amino carboxylic acid. Top trace, control; this area, we have determined the changes in CFTR distribu- bottom trace, pretreated with lovastatin (50 mM,12h). B, mean I sc responses of Calu-3 cells following pre-treatment with no drug (closed tion in polarized airway epithelial cells when the functions of circles), 50 mM lovastatin (open circles), or 50 mM lovastatin plus 2.5 mM multiple Rab proteins are disrupted by incubation with lovas- mevalonate (closed squares), 10 mM GG-PP (open squares), or 10 mM tatin, which acts to inhibit synthesis of farnesyl and gera- F-PP (closed triangles). Values represent the means 6 S.E. (n 5 5–12). nylgeranyl. First, we established that the dose of lovastatin NYS and N, nystatin; F, forskolin; CON, control; D, diphenyl amino ras carboxylic acid. used (50 mM, 12 h) inhibited farnesylation of p21 . We then showed that this dose inhibited isoproterenol-induced I across sc thionine in nonpolar cells transfected with CFTR cDNA and in HTE with a t1 of ;5 h. This was not a nonspecific toxic effect ⁄2 epithelial cells naturally expressing CFTR (24, 25). The re- because I recovered with a t1 of ;12 h, R was not altered, sc ⁄2 te maining 75% of CFTR protein is believed to be degraded in the and similar effects were obtained by treating cells with N- ER (24, 26). By contrast, essentially all DF508 CFTR is de- acetyl-S-geranylgeranyl-L-cysteine, a specific blocker of methyl graded in the ER (25, 27–29), a defect in trafficking that is esterification of geranyl geranylated proteins (Ref. 39 and data probably due to altered folding of the mutant protein (27). not shown). Interestingly, the fractions of wild-type and mutant protein Transepithelial chloride secretion (measured as isoprotere- passing from the ER to the Golgi can be altered pharmacolog- nol-induced I ) reflects the coordinated function of several sc ically. In nonpolar human airway epithelial cell lines, stimula- transport proteins in both the apical and basolateral mem- tion of the heteromeric G protein, Ga , reduced the amount of branes (40), and changes in net chloride secretion need not 1–2 wild-type CFTR trafficking to the apical membrane (30). Con- reflect changes in CFTR content of the apical membrane. versely, inhibition of Ga caused the appearance of a cAMP- Therefore, we measured CFTR activity as the temperature- 1–2 activated chloride conductance in the membranes of CF cell induced increases in I efflux. Iodide is not transported by the lines (30). basolateral NaK Cl cotransporter (41, 42) and exits cells only There is also evidence that CFTR is trafficked to and from through chloride channels. Iodide efflux from HTE does not the apical membrane by exo- and endocytosis. First, endosomes respond to elevation of cAMP (15, 16), probably because CFTR contain functional CFTR (31–34). Second, forskolin-induced is activated under base-line conditions in these cells. However, 125 125 increases in I efflux and transepithelial chloride secretion in the temperature-dependent increase in I efflux is inhibited T cells are reduced by inhibitors of microtubule formation by diphenyl amino carboxylic acid and blockers of protein ki- (35). Third, Prince et al. (36) have shown that CFTR is rapidly nase A but not by DIDS or 1,2-bis(2-aminophenoxyl) ethane- internalized in T cells or in CFPAC cells transfected with N,N,N9,N9-tetraacetic acid-acetoxymethyl ester. Thus, iodide flux from these cells is via CFTR rather than calcium-activated wild-type CFTR and that the rate of internalization is inhibited by forskolin. Of interest was the finding that the inhibitory halide channels (16). When the basolateral membrane of effect of forskolin on CFTR internalization required passage of Calu-3 cells is permeabilized with nystatin in the presence of a chloride through CFTR; it was not seen in chloride-free (gluco- transepithelial chloride gradient, then changes in I and con- sc nate) medium or in CFPAC cells transfected with nonfunc- ductance reflect changes in the apical membrane chloride ef- tional, correctly trafficking mutant CFTR (36). flux (17). In this preparation, there is no evidence for calcium- It is almost certain that all of these various routes for traf- activated chloride channels (17), and therefore cAMP-induced ficking of CFTR involve small GTP-binding proteins (21). Most changes in I across nystatin-permeabilized Calu-3 cells pro- sc of these are Rab proteins, which are 21–25 kDa in mass and are vide another assay of CFTR function. cytosolic immediately following their manufacture. However, We found that treatment with lovastatin for 12 h reduced addition of farnesyl or geranylgeranyl moieties to C-terminal temperature-induced increases in I efflux to 42 6 10 and 29 cysteine residues leads to their association with cell mem- 6 5% of control in Calu-3 cells and HTE, respectively. When branes (37). Specifically, the isoprenylated Rab proteins bind Calu-3 cells were exposed to 50 mM lovastatin for 24 h, the I of sc 25106 Trafficking of CFTR in Human Tracheal Cells Am. J. Physiol. 263, L692–L707 nystatin-permeabilized cells in the presence of forskolin was 4. Hanrahan, J. W., Tabcharani, J. A., and Grygoczyk, R. (1993) in Current 23% of control. Detailed time courses for the effects of lovasta- Topics in Cystic Fibrosis (Dodge, J. A., Brock, J. H., and Widdicombe, J. H., eds) Vol. 1, pp. 93–137, John Wiley & Sons, Inc., New York tin on CFTR function were not obtained in these studies. How- 5. Anderson, M. P., Gregory, R. J., Thompson, S., Souza, D. W., Paul, S., ever, assuming an exponential decline toward zero, these data Mulligan, R. C., Smith, A. E., and Welsh, M. J. (1991) Science. 253, 202–207 suggest that with vesicular trafficking inhibited, the t1 for 2 6. Tsui, L. C. (1992) Trends Genet. 8, 392–398 7. Welsh, M. J., Anderson, M. P., Rich, D. P., Berger, H. A., Denning, G. M., CFTR in the apical membrane is 7–11 h, which is in reasonable Ostedgaard, L. S., Sheppard, D. N., Cheng, S. H., Gregory, R. J., and Smith, agreement with the estimated turnover of ;14 h determined A. E. (1992) Neuron 8, 821–829 for CFTR in T cells treated with antisense oligonucleotides to 8. Kartner, N., Augustinas, O., Jensen, T. J., Naismith, A. L., and Riordan, J. R. (1992) Nat. Genet. 1, 321–327 CFTR mRNA (43). All of the effects of lovastatin on CFTR 9. Denning, G. M., Anderson, M. P., Amara, J. F., Marshall, J., Smith, A. E., and function could be wholly or partially reversed by the addition of Welsh, M. J. (1992) Nature 358, 761–764 10. Sheppard, D. N., Rich, D. P., Ostedgaard, L. S., Gregory, R. J., Smith, A. E., mevalonate, GG-PP, or F-PP which allow cells to regain their and Welsh, M. J. (1993) Nature 362, 160–164 protein isoprenylation capabilities. By contrast, the addition of 11. Wandinger-Ness, A., Bennett, M. K., Antony, C., and Simons, K. (1990) J. Cell cholesterol (200 mM, 24 h), which is downstream of farnesyl and Biol. 111, 987-1000 12. Ecay, T. W., and Valentich, J. D. (1993) Am. J. Physiol. 265, C422–C431 geranylgeranyl in the metabolic pathway (Fig. 1), had no effect 13. Shen, B.-Q., Barthelson, R. A., Skach, W., Gruenert, D. C., Sigal, E., Mrsny, on the inhibitory action of lovastatin (data not shown). R. J., and Widdicombe, J. H. (1993) J. Biol. Chem. 268, 19070–19075 14. Yamaya, M., Finkbeiner, W. E., Chun, S. Y., and Widdicombe, J. H. (1992) Similar effects of lovastatin on chloride transport have been Am. J. Physiol. 262, L713–L724 reported for T cells (12). In these cells lovastatin also abol- 15. Ohrui, T., Shen, B.-Q., Mrsny, R. J., and Widdicombe, J. H. (1995) J. Appl. ished I , though at longer exposures (3 days) than those used Physiol. 78, 1197–1202 sc 16. Shen, B.-Q., Mrsny, R. J., Finkbeiner, W. E., and Widdicombe, J. H. (1995) by us. However, unlike our results, lovastatin markedly de- Am. J. Physiol., in press creased R (to zero with 10 mM lovastatin for 3 days) in those te 17. Shen, B.-Q., Finkbeiner, W. E., Wine, J. J., Mrsny, R. J., and Widdicombe, studies. Furthermore, recovery from lovastatin exposure was J. H. (1994) Am. J. Physiol. 266, L493-L501 18. Goldstein, J. L., and Brown, M. S. (1990) Nature 343, 425–430 not assessed. When function of apical membrane cAMP-acti- 19. Leonard, S., Beck, L., and Sinensky, M. (1990) J. Biol. Chem. 265, 5157–5160 vated chloride conductance was assessed in nystatin-permeabi- 20. Gutierrez, L., Magee, A. I., Marshall, C. J., and Hancock, J. F. (1989) EMBO J. 8, 1093–1098 lized T cells, exposure to 2.5 mg/ml for 2 days reduced the 21. Pfeffer, S. (1992) Trends Cell Biol. 2, 41–46 cAMP-stimulated change in I to ;50% of control (12). sc 22. Deleted in proof Alterations in the cAMP-dependent apical membrane chlo- 23. Deleted in proof 24. Cheng, S. H., Gregory, R. J., Amara, J. F., Smith, A. E., Rich, D. P., Anderson, ride conductance could reflect changes in the properties of M., and Welsh, M. J. (1993) in Cystic Fibrosis: Current Topics (Dodge, J. A., individual channels (e.g. unitary conductance or probability of Brock, D. J. H., and Widdicombe, J. H., eds) Vol. 1, pp. 175–189, Wiley & Sons, Inc., New York opening) or could be due to changes in channel density. These 25. Pind, S., Riordan, J. R., and Williams, D. B. (1994) J. Biol. Chem. 269, possibilities can only be definitively distinguished by patch- 12784–12788 clamp analysis. However, because lovastatin markedly de- 26. Yang, Y., Engelhardt, J. F., and Wilson, J. M. (1994) Am. J. Respir. Cell Mol. Biol. 11, 7–15 creased total CFTR in both Calu-3 and HTE (Fig. 7), we spec- 27. Thomas, P., Ko, Y., and Pedersen, P. (1992) FEBS Lett. 312, 7–9 ulate that inhibition of trafficking pathways resulted in 28. Yang, Y., Janich, S., Cohn, J. A., and Wilson, J. M. (1993) Proc. Natl. Acad. Sci. decreased CFTR content of the apical membrane. U. S. A. 90, 9480–9484 29. Ward, C. L., and Kopito, R. R. (1994) J. Biol. Chem. 269, 25710–25718 In summary, a variety of approaches suggest that lovastatin 30. Schwiebert, E. M., Gesek, F., Ercolani, L., Wjasow, C., Gruenert, D. C., reduces the numbers of cAMP-dependent chloride channels Karlson, K., and Stanton, B. A. (1994) Am. J. Physiol. 267, C272–C281 31. Lukacs, G. L., Chang, X. B., Kartner, N., Rotstein, O. D., Riordan, J. 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Future studies will be directed at identifying the Sci. U. S. A. 91, 5192–5196 37. Magee, T., and Newman, C. (1992) Trends Cell Biol. 2, 318–323 individual GTP-binding proteins involved in these events. 38. Glomset, J., Gelb, M., and Farnsworth, C. (1990) Trends Biochem. Sci. 15, 139–142 Acknowledgments—We thank Roger Barthelson for technical discus- 39. Volker, C., Miller, R., McCleary, W., Rao, A., Poenie, M., Backer, J., and Stock, sions and Rodney Pearlman and Tue Nguyen for support. J. (1991) J. Biol. Chem. 266, 21515–21522 40. Welsh, M. J. (1987) Physiol. Rev. 67, 1143–1184 REFERENCES 41. Widdicombe, J. H., Nathanson, I. T., and Highland, E. (1983) Am. J. Physiol. 245, C388–C396 1. Riordan, J. R., Rommens, J. M., Kerem, B., Alon, N., Rozmahel, R., Grzelczak, Z., Zielenski, J., Lok, S., Plavsic, N., Chou, J.-L., Drumm, M. L., Iannuzzi, 42. O’Grady, S. M., Palfrey, H. C., and Field, M. (1987) Am. J. Physiol. 253, C177–C192 M. C., Collins, F. S., and Tsui, L.-C. (1989) Science. 245, 1066–1073 2. Haws, C., Finkbeiner, W. E., Widdicombe, J. H., and Wine, J. J. (1994) Am. J. 43. Wagner, J. A., McDonald, T. V., Nghiem, P. T., Lowe, A. W., Schulman, H., Gruenert, D. C., Stryer, L., and Gardner, P. (1992) Proc. Natl. Acad. Sci. Physiol. 266, L502–L512 3. Haws, C., Krouse, M. E., Xia, Y., Gruenert, D. C., and Wine, J. J. (1992) U. S. A. 89, 6785–6789 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Effects of Lovastatin on Trafficking of Cystic Fibrosis Transmembrane Conductance Regulator in Human Tracheal Epithelium

Shen, Ben-Quan; Widdicombe, Jonathan H.; Mrsny, Randall J.
Journal of Biological Chemistry – Oct 1, 1995
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THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 270, No. 42, Issue of October 20, pp. 25102–25106, 1995 © 1995 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Effects of Lovastatin on Trafficking of Cystic Fibrosis Transmembrane Conductance Regulator in Human Tracheal Epithelium* (Received for publication, July 13, 1995) Ben-Quan Shen‡, Jonathan H. Widdicombe‡, and Randall J. Mrsny§ From the ‡Cardiovascular Research Institute, University of California, San Francisco, California 94143-0130, the ‡Children’s Hospital Oakland Research Institute, Oakland, California 94609, and the ‡§Department of Pharmaceutical Research and Development, Genentech, Inc., South San Francisco, California 94080-4980 residue at the 508 position (DF508) (6). In transfected cells, Genetic defects in the cystic fibrosis transmembrane conductance regulator (CFTR), a cAMP-activated chlo- many of these CF gene mutations, including the DF508 variant, ride channel, cause cystic fibrosis. Most defective forms result in protein products that are not completely processed, of CFTR show improper intracellular trafficking. Be- are retained in the ER, and are trafficked into a degradation cause isoprenylated, small GTP-binding proteins are in- pathway associated with the ER rather than to the plasma volved in the vesicular trafficking of other integral membrane (7, 8). At reduced temperatures (,30 °C), CFTR membrane proteins, we have investigated the role of DF508 and other mutant forms are correctly trafficked to the isoprenylation in the trafficking of CFTR to the apical plasma membrane and confer a cAMP-activated chloride con- membranes of primary cultures of human airway epi- ductance of similar magnitude to that produced by wild-type thelium and of Calu-3 cells, a human lung carcinoma cell CFTR (9). Less frequently observed defective forms of CFTR line. CFTR function was measured as short circuit cur- are trafficked properly to the apical plasma membrane at 37 °C rent, I efflux, and conductance of cell sheets with but show reduced capacity as chloride channels (10). permeabilized basolateral membranes. Lovastatin, an Little is known about the trafficking pathways for wild-type inhibitor of isoprenyl lipid biosynthesis, markedly in- CFTR. Previous studies have demonstrated that a class of hibited all measures of CFTR function. The lovastatin- isoprenylated small GTP-binding proteins (termed Rab pro- induced declines in CFTR function were corrected by teins) are involved in the trafficking of intracellular vesicles the simultaneous addition of mevalonate or the isopre- (such as those that would contain CFTR) to the plasma mem- nyl lipids geranylgeranyl and farnesyl but not choles- brane of polarized epithelial cells (11). A recent report suggests terol. Lovastatin reduced total cellular CFTR as that isoprenylated proteins may be involved in trafficking of assessed by immunoprecipitation. Mevalonate or CFTR in a colonic carcinoma cell line (12). The results reported isoprenyl lipids protected CFTR levels from the actions of lovastatin. Together, these results suggest a role for here strongly support a role for isoprenylated proteins in the isoprenyl lipids, presumably through the actions of trafficking of the CFTR to the apical membrane of human small GTP-binding proteins, in the trafficking of CFTR airway epithelium. to the apical membrane of human airway epithelium. MATERIALS AND METHODS Cell Culture—Primary cultures of human tracheal epithelium (HTE) or a human lung carcinoma cell line (Calu-3) were plated at 10 cells/ Cystic fibrosis (CF) is the commonest lethal genetic disease cm and grown to confluency on Transwellt inserts (pore size, 0.45 mm; in Caucasians. The gene responsible for CF has been identified 2 surface area, 1 cm ; Costar, Cambridge, MA) coated with human pla- (1) and found to encode a 170-kDa glycoprotein known as the cental collagen (13). High levels of differentiation of HTE were achieved cystic fibrosis transmembrane conductance regulator (CFTR). by adding media containing 2% Ultraser G to the basolateral side of the insert only, leaving an air-interface at the apical surface (14). HTE Several lines of evidence have suggested that CFTR is a cAMP- cultures were used once the transepithelial resistance (R ) was .250 regulated chloride channel in the apical membrane of airway te ohmszcm , which occurred approximately 5–7 days after plating. Calu-3 (2, 3) and other epithelial cells (4). A relationship between cells were used once a R of .100 ohmszcm was achieved, typically te mutations in the CF gene and defective chloride conductance after 10–14 days of culture. has been clearly demonstrated (5). Additions of lovastatin (Merck Sharp & Dohme) were made from a Although a variety of mutations in the CFTR gene lead to 50-fold concentrated ethanolic solution to produce a final concentration of 50 mM. Mevalonic acid (Sigma), farnesyl pyrophosphate (F-PP), and CF, the commonest involves a deletion of the phenylalanine geranylgeranyl pyrophosphate (GG-PP) (American Radiolabeled Chem- icals; St. Louis, MO) were added from stocks prepared in 70% ethanol * This work was supported by a grant from Cystic Fibrosis Research and 0.075 M NH HCO . Cholesterol (Sigma) was added from an ether 4 3 Inc. (to B.-Q. S.) and by National Institutes of Health Specialized stock. The same volumes of vehicles alone were added to control cells. Center of Research Grant HL-42368. The costs of publication of this Assessment of Ras Isoprenylation—Cells were harvested in Tris- article were defrayed in part by the payment of page charges. This buffered saline (150 mM NaCl and 50 mM Tris-HCl, pH 8.0) containing article must therefore be hereby marked “advertisement” in accordance 1% Triton X-100, 0.1% SDS, 1% sodium deoxycholate, and 1 mM phen- with 18 U.S.C. Section 1734 solely to indicate this fact. ylmethylsulfonyl fluoride. Lysate (200 mg protein in 100 ml) was mixed To whom correspondence should be addressed: Genentech, Inc., with an equal volume of 2 3 SDS sample buffer (1 3 sample buffer Mailstop #6, 460 Pt. San Bruno Blvd., South San Francisco, CA 94080- consisted of 10% glycerol, 62.5 mM Tris-HCl, pH 6.8, 5% b-mercapto- 4980. Tel.: 415-225-2592; Fax: 415-225-1418. ethanol, 2% SDS, and 0.002% bromphenol blue). Following incubation The abbreviations used are: CF, cystic fibrosis; CFTR, cystic fibrosis at 100 °C for 3 min, protein components were separated by SDS-PAGE transmembrane regulator; DIDS, 4,49-diisothiocyanostilbene-2,29-di- (10–18% gel) and transferred onto a nitrocellulose membrane sulfonic acid; ER, endoplasmic reticulum; F-PP, farnesyl pyrophos- (Schleicher & Schuell) at 4 °C for 60 min in 25 mM Tris-HCl, 150 mM phate; GG-PP, geranylgeranyl pyrophosphate; HTE, human tracheal epithelium; I , short circuit current; PAGE, polyacrylamide gel electro- glycine, 0.05% SDS, and 10% methanol, pH 8.3. After transfer, mem- sc phoresis; R , transepithelial resistance. branes were blocked in 1% nonfat dried milk, 1% bovine serum albu- te This is an Open Access article under the CC BY license. Trafficking of CFTR in Human Tracheal Cells 25103 min, 1% polyvinylpyrolidone 10, and 10 mM Na EDTA in Tris-buffered saline overnight at 4 °C. After two 5-min washes in Tris-buffered saline containing 0.05% Tween 20, the membrane was washed twice in wash buffer (1% nonfat dried milk, 0.5% bovine serum albumin and 0.05% Tween 20 in Tris-buffered saline). The membrane was incubated with 10 mg/ml primary antibody (pan-Ras (Ab-3) mouse monoclonal; Onco- gene Science Inc., Manhasset, NY) in wash buffer at room temperature for 2 h, washed 4 times in wash buffer, and incubated at room temper- ature for 1.5 h in wash buffer containing a goat anti-mouse IgG horse- radish peroxidase-conjugated secondary antibody (Zymed Labs, Inc., South San Francisco, CA) diluted 1:2,000 in wash buffer. After 4 washes with wash buffer, Ras proteins were detected with an enhanced chemi- luminescence kit (Amersham Corp). Measurement of Short Circuit Current (I ) and Transepithelial Re- sc sistance (R )—Cell sheets were mounted in Ussing chambers and te bathed with bicarbonate-buffered Krebs-Henseleit solution (pH 7.4) mixed by a gas lift of 95% O and 5% CO at 37 °C. Transepithelial 2 2 potential differences were clamped to zero, and the resulting I was sc displayed continuously on a pen recorder. Every 20 s the R was te determined from the size of the current deflections resulting from 0.2-s voltage pulses of constant amplitude (0.2–1 mV). Stimulation of cAMP- dependent chloride secretion was achieved by the addition of 10 M FIG.1. Diagram of the dedicated cholesterol synthetic path- isoproterenol or 10 M forskolin. way. Note the location of lovastatin inhibition of 3-hydroxy-3-methyl- Halide Efflux—Effluxes were performed as described (15, 16). Cell glutaryl coenzyme A reductase in the pathway. Also note that the sheets were loaded with I by placing them in serum-free medium 125 pathway intermediates, mevalonate, F-PP, and GG-PP come after the containing Na I (10 mCi/ml, 3 nM, and 17.4 Ci/mg; DuPont NEN) for point of lovastatin inhibition. PP, pyrophospate. 2 h. Residual surface-associated tracer was removed with two 200-ml washes (15 s each) using Krebs-Henseleit solution, and monolayers were placed, mucosal side up, in the top half of a Swinnex 25-mm filter holder (Millipore Corp. Bedford, MA) containing a 0.65-mm pore, type D, cellulose ester filter. Oxygenated Krebs-Henseleit solution was passed over the mucosal surface of the monolayers and through surrounding pores in the filter holder at 1 ml/min using a peristaltic pump. Effluent fractions were collected at 1-min intervals. The perfusate temperature was raised to 37 °C using a heated copper wire wrapped around the glass inlet tubing. This wire connected to a temperature controller, which responded to a thermocouple inside the Swinnex filter holder. ras FIG.2. Lovastatin alters the mobility of p21 in SDS-PAGE. Stable base-line efflux was achieved after an initial 7-min period at HTE were exposed for 12 h to vehicle alone (lane 1), 50 mM lovastatin room temperature. Over the next 2 min, perfusion temperature was (lane 2), or 50 mM lovastatin plus 2.5 mM mevalonate (lane 3). Levels of raised to 37 °C. We have shown elsewhere (16) that the temperature- ras p21 were determined by conventional Western blotting. Locations of induced increase in I efflux occurs via CFTR. We collected 13 further ras ras the cytosolic (c-p21 ) and membrane-associated (m-p21 ) forms of effluent samples after the initial seven. All samples were counted on a ras p21 are denoted. g 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 ras the addition of 50 mM lovastatin to HTE, a retardation of p21 counts lost/min of sampling period divided by the average total counts electrophoretic mobility in SDS-PAGE was seen in Western present in the cells during that time period). blots (Fig. 2), reflecting a reduction in the levels of membrane- Nystatin Permeabilization—Cell sheets were bathed with a Krebs- associated Ras and an accumulation of the cytosolic form as Henseleit solution on their serosal side (120 mM NaCl) and on their mucosal sides with a solution in which all but 20 mM NaCl was replaced described previously (19). These results show that these condi- by sodium gluconate. Nystatin (0.72 mg/ml; Sigma) was added to the tions of lovastatin exposure resulted in a reduction in the serosal bath to permeabilize the basolateral membranes. Under these extent of isoprenylation of HTE. Similar results were obtained circumstances, changes in R and I reflect apical membrane chloride te sc on Calu-3 cells (data not shown). Mevalonate is a pathway channel activity (17). intermediate whose production is blocked by lovastatin. When Immunoprecipitation of CFTR—Our methods are described in full added at 2.5 mM at the same time as lovastatin, conversion of elsewhere (13). In brief, cell sheets were lysed with 0.1% SDS, Triton ras X-100, and sodium deoxycholate; samples of lysate were incubated with the cytosolic to the membrane-associated form of p21 was a mouse monoclonal antibody raised against a C terminus peptide of restored (Fig. 2). human CFTR (Genzyme, Cambridge, MA). Antibody-antigen complexes Ussing chamber studies provided an initial assessment of were then adsorbed onto pansorbin cells (Calbiochem) and pelleted. the effects of lovastatin on chloride secretion. Base-line short CFTR was then phosphorylated by adding protein kinase A catalytic circuit current (I ) of HTE was ;5 mA/cm but reached 15–20 subunit (Sigma) and [g- P]ATP (30 Ci/mmol; 2 mCi/ml; DuPont NEN). sc mA/cm after stimulation with 10 mM isoproterenol, an increase Phosphoproteins were separated by 6% SDS-PAGE, and levels of P were determined by densitometric scans of autoradiographs. reflecting induction of chloride secretion (14). Treatment with 50 mM lovastatin completely blocked the chloride secretion in- RESULTS duced by isoproterenol after approximately 12 h of exposure Lovastatin, an inhibitor of 3-hydroxy-3-methylglutaryl coen- (Fig. 3). Washout of the lovastatin resulted in complete recov- zyme A reductase, blocks one of the earliest dedicated steps of ery of the isoproterenol-induced increase in I after a further sc the cholesterol synthetic pathway and inhibits the production 24 h of incubation (Fig. 3). Lovastatin at all exposure times had of several metabolically important intermediates (18) (Fig. 1). no significant effect on R (data not shown). te In order to determine whether lovastatin inhibited isoprenyla- HTE treated for 12 h with 50 mM lovastatin showed reduced tion of proteins in our cells, our first studies investigated the increases in I efflux in response to a temperature change ras extent to which post-translational modification of p21 had from 22 to 37 °C (Fig. 4A). To evaluate the relative importance ras occurred. The primary translation product of p21 is cytosolic. of reduced levels of the 15-carbon farnesyl and 20-carbon gera- On isoprenylation it becomes membrane-bound and shows nylgeranyl isoprenoid intermediates of the cholesterol syn- slightly increased mobility on SDS-PAGE (19, 20). 12 h after thetic pathways, the pyrophosphate forms of these intermedi- 25104 Trafficking of CFTR in Human Tracheal Cells FIG.3. Lovastatin inhibits isoproterenol-induced chloride se- cretion. I responses to 10 mM isoproterenol are shown for HTE treated sc with 50 mM lovastatin (filled circles), untreated time controls (open circles), and cells recovering from a 24 h exposure to lovastatin (trian- gles). The means 6 S.E. are shown (n 5 4–5). FIG.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 6 S.E. (n 5 5). block the inhibition induced by lovastatin (data not shown). 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 ap- ical membrane (17). In the presence of a transepithelial chlo- ride gradient, I of Calu-3 cells increased on exposure to nys- sc tatin (Fig. 6), reflecting the formation of a chloride concentration gradient across the apical membrane. Further stimulation of I occurred following the addition of forskolin. sc This stimulation of I was inhibited by the addition of 2 mM sc diphenyl amino carboxylic acid, a blocker of the CFTR chloride conductance (4). Pretreatment of Calu-3 cells with 50 mM lov- astatin dramatically inhibited the increases in I associated sc 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 mM GG-PP was slightly less protective, and 10 mM F-PP offered only a moderate amount of protection (Fig. 6). Disruption of the cholesterol synthetic pathway by lovastatin has previously been shown to impede intracellular trafficking by removing the farnesyl and geranylgeranyl lipids required FIG.4. Lovastatin inhibits temperature-induced I efflux for effective membrane association of small GTP-binding pro- from HTE. A, effluxes of I were measured using confluent cell sheets teins (21). Because treatment with lovastatin resulted in a isolated from the same trachea. Cells were exposed to control buffer significant decrease in the cAMP-dependent iodide efflux from (open circles), 50 mM lovastatin (filled circles), 50 mM lovastatin plus 2.5 Calu-3 cells, it was not surprising that lovastatin (50 mM,24h) mM mevalonate (open squares), 50 mM lovastatin plus 10 mM GG-PP (filled squares), or 50 mM lovastatin plus 10 mM F-PP (filled triangles). reduced total CFTR levels of Calu-3 cells to 40 6 7% of control, Temperature was increased from 22 to 37 °C as indicated by the hori- as determined by immunoprecipitation (Fig. 7). The observed zontal bar. B, increases in efflux between 8 and 11 min. The means 6 net reduction of cellular CFTR content under conditions in- S.E. are shown (n 5 5). CONT, control; LOVA, lovastatin; L 1 M, duced by lovastatin suggests that the degradation route(s) for lovastatin plus mevalonate; L 1 G, lovastatin plus GG-PP; L 1 F, lovastatin plus F-PP; *, significantly different from control; †, signifi- this chloride channel is still competent. The effect of lovastatin cantly different from lovastatin alone. on total CFTR content was reversed by 2.5 mM mevalonate (Fig. 7), 10 mM GG-PP, or 10 mM F-PP (data not shown). ates (F-PP and GG-PP) were added to cells at the same time as 125 DISCUSSION lovastatin. Comparison of the increase in I efflux between the time of temperature change (8 min) and the maximal in- Trafficking pathways for CFTR are rapidly being elucidated. crease (11 min) demonstrated that cells simultaneously ex- CFTR is synthesized and partially glycosylated in the ER. posed to lovastatin plus 2.5 mM mevalonate, 10 mM F-PP, or 10 During passage through the Golgi, the protein becomes fully mM GG-PP were protected from the negative effects of lovasta- glycosylated and is transported to the apical membrane. How- tin (Fig. 4B). Similar results were obtained using Calu-3 cell ever, only ;25% of wild-type CFTR successfully passes from monolayers (Fig. 5). By contrast, 250 mM cholesterol did not the ER to the Golgi as revealed by pulse-labeling with [ S]me- Trafficking of CFTR in Human Tracheal Cells 25105 FIG.7. Lovastatin treatment reduces total cellular CFTR con- tent. 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 mM,12h). Lane 4, cells treated with lovas- tatin and mevalonate (2.5 mM). 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 re- ceptors on the target membrane, leading to membrane fusion. Conversion of GTP to GDP leads to the return of the Rab FIG.6. Lovastatin inhibits forskolin-induced I responses in sc nystatin-permeabilized cell sheets. A, typical I tracing of conflu- protein to the cytosol. sc ent monolayers of Calu-3 cells measured in Ussing chambers. After a A knowledge of which GTP-binding proteins regulate the stable base line was achieved, 0.72 mg/ml nystatin was added to the various pathways for CFTR movement within airway epithelial serosal bath. After another 15 min, 10 M forskolin was added, fol- cells could lead to therapies for CF. As an initial approach to lowed by 2 mM diphenyl amino carboxylic acid. Top trace, control; this area, we have determined the changes in CFTR distribu- bottom trace, pretreated with lovastatin (50 mM,12h). B, mean I sc responses of Calu-3 cells following pre-treatment with no drug (closed tion in polarized airway epithelial cells when the functions of circles), 50 mM lovastatin (open circles), or 50 mM lovastatin plus 2.5 mM multiple Rab proteins are disrupted by incubation with lovas- mevalonate (closed squares), 10 mM GG-PP (open squares), or 10 mM tatin, which acts to inhibit synthesis of farnesyl and gera- F-PP (closed triangles). Values represent the means 6 S.E. (n 5 5–12). nylgeranyl. First, we established that the dose of lovastatin NYS and N, nystatin; F, forskolin; CON, control; D, diphenyl amino ras carboxylic acid. used (50 mM, 12 h) inhibited farnesylation of p21 . We then showed that this dose inhibited isoproterenol-induced I across sc thionine in nonpolar cells transfected with CFTR cDNA and in HTE with a t1 of ;5 h. This was not a nonspecific toxic effect ⁄2 epithelial cells naturally expressing CFTR (24, 25). The re- because I recovered with a t1 of ;12 h, R was not altered, sc ⁄2 te maining 75% of CFTR protein is believed to be degraded in the and similar effects were obtained by treating cells with N- ER (24, 26). By contrast, essentially all DF508 CFTR is de- acetyl-S-geranylgeranyl-L-cysteine, a specific blocker of methyl graded in the ER (25, 27–29), a defect in trafficking that is esterification of geranyl geranylated proteins (Ref. 39 and data probably due to altered folding of the mutant protein (27). not shown). Interestingly, the fractions of wild-type and mutant protein Transepithelial chloride secretion (measured as isoprotere- passing from the ER to the Golgi can be altered pharmacolog- nol-induced I ) reflects the coordinated function of several sc ically. In nonpolar human airway epithelial cell lines, stimula- transport proteins in both the apical and basolateral mem- tion of the heteromeric G protein, Ga , reduced the amount of branes (40), and changes in net chloride secretion need not 1–2 wild-type CFTR trafficking to the apical membrane (30). Con- reflect changes in CFTR content of the apical membrane. versely, inhibition of Ga caused the appearance of a cAMP- Therefore, we measured CFTR activity as the temperature- 1–2 activated chloride conductance in the membranes of CF cell induced increases in I efflux. Iodide is not transported by the lines (30). basolateral NaK Cl cotransporter (41, 42) and exits cells only There is also evidence that CFTR is trafficked to and from through chloride channels. Iodide efflux from HTE does not the apical membrane by exo- and endocytosis. First, endosomes respond to elevation of cAMP (15, 16), probably because CFTR contain functional CFTR (31–34). Second, forskolin-induced is activated under base-line conditions in these cells. However, 125 125 increases in I efflux and transepithelial chloride secretion in the temperature-dependent increase in I efflux is inhibited T cells are reduced by inhibitors of microtubule formation by diphenyl amino carboxylic acid and blockers of protein ki- (35). Third, Prince et al. (36) have shown that CFTR is rapidly nase A but not by DIDS or 1,2-bis(2-aminophenoxyl) ethane- internalized in T cells or in CFPAC cells transfected with N,N,N9,N9-tetraacetic acid-acetoxymethyl ester. Thus, iodide flux from these cells is via CFTR rather than calcium-activated wild-type CFTR and that the rate of internalization is inhibited by forskolin. Of interest was the finding that the inhibitory halide channels (16). When the basolateral membrane of effect of forskolin on CFTR internalization required passage of Calu-3 cells is permeabilized with nystatin in the presence of a chloride through CFTR; it was not seen in chloride-free (gluco- transepithelial chloride gradient, then changes in I and con- sc nate) medium or in CFPAC cells transfected with nonfunc- ductance reflect changes in the apical membrane chloride ef- tional, correctly trafficking mutant CFTR (36). flux (17). In this preparation, there is no evidence for calcium- It is almost certain that all of these various routes for traf- activated chloride channels (17), and therefore cAMP-induced ficking of CFTR involve small GTP-binding proteins (21). Most changes in I across nystatin-permeabilized Calu-3 cells pro- sc of these are Rab proteins, which are 21–25 kDa in mass and are vide another assay of CFTR function. cytosolic immediately following their manufacture. However, We found that treatment with lovastatin for 12 h reduced addition of farnesyl or geranylgeranyl moieties to C-terminal temperature-induced increases in I efflux to 42 6 10 and 29 cysteine residues leads to their association with cell mem- 6 5% of control in Calu-3 cells and HTE, respectively. When branes (37). Specifically, the isoprenylated Rab proteins bind Calu-3 cells were exposed to 50 mM lovastatin for 24 h, the I of sc 25106 Trafficking of CFTR in Human Tracheal Cells Am. J. Physiol. 263, L692–L707 nystatin-permeabilized cells in the presence of forskolin was 4. Hanrahan, J. W., Tabcharani, J. A., and Grygoczyk, R. (1993) in Current 23% of control. Detailed time courses for the effects of lovasta- Topics in Cystic Fibrosis (Dodge, J. A., Brock, J. H., and Widdicombe, J. H., eds) Vol. 1, pp. 93–137, John Wiley & Sons, Inc., New York tin on CFTR function were not obtained in these studies. How- 5. Anderson, M. P., Gregory, R. J., Thompson, S., Souza, D. W., Paul, S., ever, assuming an exponential decline toward zero, these data Mulligan, R. C., Smith, A. E., and Welsh, M. J. (1991) Science. 253, 202–207 suggest that with vesicular trafficking inhibited, the t1 for 2 6. Tsui, L. C. (1992) Trends Genet. 8, 392–398 7. Welsh, M. J., Anderson, M. P., Rich, D. P., Berger, H. A., Denning, G. M., CFTR in the apical membrane is 7–11 h, which is in reasonable Ostedgaard, L. S., Sheppard, D. N., Cheng, S. H., Gregory, R. J., and Smith, agreement with the estimated turnover of ;14 h determined A. E. (1992) Neuron 8, 821–829 for CFTR in T cells treated with antisense oligonucleotides to 8. Kartner, N., Augustinas, O., Jensen, T. J., Naismith, A. L., and Riordan, J. R. (1992) Nat. Genet. 1, 321–327 CFTR mRNA (43). All of the effects of lovastatin on CFTR 9. Denning, G. M., Anderson, M. P., Amara, J. F., Marshall, J., Smith, A. E., and function could be wholly or partially reversed by the addition of Welsh, M. J. (1992) Nature 358, 761–764 10. Sheppard, D. N., Rich, D. P., Ostedgaard, L. S., Gregory, R. J., Smith, A. E., mevalonate, GG-PP, or F-PP which allow cells to regain their and Welsh, M. J. (1993) Nature 362, 160–164 protein isoprenylation capabilities. By contrast, the addition of 11. Wandinger-Ness, A., Bennett, M. K., Antony, C., and Simons, K. (1990) J. Cell cholesterol (200 mM, 24 h), which is downstream of farnesyl and Biol. 111, 987-1000 12. Ecay, T. W., and Valentich, J. D. (1993) Am. J. Physiol. 265, C422–C431 geranylgeranyl in the metabolic pathway (Fig. 1), had no effect 13. Shen, B.-Q., Barthelson, R. A., Skach, W., Gruenert, D. C., Sigal, E., Mrsny, on the inhibitory action of lovastatin (data not shown). R. J., and Widdicombe, J. H. (1993) J. Biol. Chem. 268, 19070–19075 14. Yamaya, M., Finkbeiner, W. E., Chun, S. Y., and Widdicombe, J. H. (1992) Similar effects of lovastatin on chloride transport have been Am. J. Physiol. 262, L713–L724 reported for T cells (12). In these cells lovastatin also abol- 15. Ohrui, T., Shen, B.-Q., Mrsny, R. J., and Widdicombe, J. H. (1995) J. Appl. ished I , though at longer exposures (3 days) than those used Physiol. 78, 1197–1202 sc 16. Shen, B.-Q., Mrsny, R. J., Finkbeiner, W. E., and Widdicombe, J. H. (1995) by us. However, unlike our results, lovastatin markedly de- Am. J. Physiol., in press creased R (to zero with 10 mM lovastatin for 3 days) in those te 17. Shen, B.-Q., Finkbeiner, W. E., Wine, J. J., Mrsny, R. J., and Widdicombe, studies. Furthermore, recovery from lovastatin exposure was J. H. (1994) Am. J. Physiol. 266, L493-L501 18. Goldstein, J. L., and Brown, M. S. (1990) Nature 343, 425–430 not assessed. When function of apical membrane cAMP-acti- 19. Leonard, S., Beck, L., and Sinensky, M. (1990) J. Biol. Chem. 265, 5157–5160 vated chloride conductance was assessed in nystatin-permeabi- 20. Gutierrez, L., Magee, A. I., Marshall, C. J., and Hancock, J. F. (1989) EMBO J. 8, 1093–1098 lized T cells, exposure to 2.5 mg/ml for 2 days reduced the 21. Pfeffer, S. (1992) Trends Cell Biol. 2, 41–46 cAMP-stimulated change in I to ;50% of control (12). sc 22. Deleted in proof Alterations in the cAMP-dependent apical membrane chlo- 23. Deleted in proof 24. Cheng, S. H., Gregory, R. J., Amara, J. F., Smith, A. E., Rich, D. P., Anderson, ride conductance could reflect changes in the properties of M., and Welsh, M. J. (1993) in Cystic Fibrosis: Current Topics (Dodge, J. A., individual channels (e.g. unitary conductance or probability of Brock, D. J. H., and Widdicombe, J. H., eds) Vol. 1, pp. 175–189, Wiley & Sons, Inc., New York opening) or could be due to changes in channel density. These 25. Pind, S., Riordan, J. R., and Williams, D. B. (1994) J. Biol. Chem. 269, possibilities can only be definitively distinguished by patch- 12784–12788 clamp analysis. However, because lovastatin markedly de- 26. Yang, Y., Engelhardt, J. F., and Wilson, J. M. (1994) Am. J. Respir. Cell Mol. Biol. 11, 7–15 creased total CFTR in both Calu-3 and HTE (Fig. 7), we spec- 27. Thomas, P., Ko, Y., and Pedersen, P. (1992) FEBS Lett. 312, 7–9 ulate that inhibition of trafficking pathways resulted in 28. Yang, Y., Janich, S., Cohn, J. A., and Wilson, J. M. (1993) Proc. Natl. Acad. Sci. decreased CFTR content of the apical membrane. U. S. A. 90, 9480–9484 29. Ward, C. L., and Kopito, R. R. (1994) J. Biol. Chem. 269, 25710–25718 In summary, a variety of approaches suggest that lovastatin 30. Schwiebert, E. M., Gesek, F., Ercolani, L., Wjasow, C., Gruenert, D. C., reduces the numbers of cAMP-dependent chloride channels Karlson, K., and Stanton, B. A. (1994) Am. J. Physiol. 267, C272–C281 31. Lukacs, G. L., Chang, X. B., Kartner, N., Rotstein, O. D., Riordan, J. R., and (CFTR) in the apical plasma membrane of human airway cells. Grinstein, S. (1992) J. Biol. Chem. 267, 14568–14572 These effects could be reversed by mevalonate, farnesyl, or 32. Biwersi, J., and Verkman, A. S. (1994) Am J. Physiol. 266, C149–C156 geranylgeranyl (intermediates of the cholesterol synthetic 33. Bradbury, N. A., Cohn, J. A., Venglarik, C. J., and Bridges, R. J. (1994) J. Biol. Chem. 269, 8296–8302 pathway) but not by cholesterol. The most likely explanation 34. Webster, P., Vanacore, L., Nairn, A. C., and Marino, C. R. (1994) Am. J. for these results is that lovastatin lowers levels of isoprenyl Physiol. 267, C340–C348 35. Fuller, C. M., Bridges, R. J., and Benos, D. J. (1994) Am. J. Physiol. 266, lipids, thereby inhibiting the function of small GTP-binding C661–C668 proteins and disrupting the trafficking of CFTR to the apical 36. Prince, L. S., Workman, R., Jr., and Marchase, R. B. (1994) Proc. Natl. Acad. membrane. 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