Antisense oligonucleotide to PKC-epsilon alters cAMP-dependent stimulation of CFTR in Calu-3 cells

Carole M. Liedtke and Thomas S. Cole

Cystic Fibrosis Center and Departments of Pediatrics and Physiology and Biophysics, Case Western Reserve University, Cleveland, Ohio 44106

    ABSTRACT
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Protein kinase C (PKC) regulates cystic fibrosis transmembrane conductance regulator (CFTR) channel activity but the PKC signaling mechanism is not yet known. The goal of these studies was to identify PKC isotype(s) required for control of CFTR function. CFTR activity was measured as 36Cl efflux in a Chinese hamster ovary cell line stably expressing wild-type CFTR (CHO-wtCFTR) and in a Calu-3 cell line. Chelerythrine, a PKC inhibitor, delayed increased CFTR activity induced with phorbol 12-myristate 13-acetate or with the cAMP-generating agents (-)-epinephrine or forskolin plus 8-(4-chlorophenylthio)adenosine 3',5'- cyclic monophosphate. Immunoblot analysis of Calu-3 cells revealed that PKC-alpha , -beta II, -delta , -epsilon , and -zeta were expressed in confluent cell cultures. Pretreatment of cell monolayers with Lipofectin plus antisense oligonucleotide to PKC-epsilon for 48 h prevented stimulation of CFTR with (-)-epinephrine, reduced PKC-epsilon activity in unstimulated cells by 52.1%, and decreased PKC-epsilon mass by 76.1% but did not affect hormone-activated protein kinase A activity. Sense oligonucleotide to PKC-epsilon and antisense oligonucleotide to PKC-delta and -zeta did not alter (-)-epinephrine-stimulated CFTR activity. These results demonstrate the selective regulation of CFTR function by constitutively active PKC-epsilon .

Chinese hamster ovary cell line; chelerythrine; chloride efflux; epinephrine; protein kinase A; protein kinase C; cystic fibrosis transmembrane conductance regulator

    INTRODUCTION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

CYSTIC FIBROSIS (CF) is a disease of electrolyte transport abnormalities that is characterized by the production of dehydrated viscous secretions in lungs, pancreatic duct, and intestinal tract. The genetic basis for CF is mutation of the CF transmembrane conductance regulator (CFTR), a secretory Cl channel and conductance regulator. CFTR is a 1,480-amino acid protein with a unique structure characterized by three cytoplasmic domains, two nucleotide binding folds, and a regulatory (R) domain that contains consensus sequences for phosphorylation by protein kinase A (PKA) and by protein kinase C (PKC). There is direct evidence for phosphorylation of serine residues in the R domain by PKA (5, 24) and by PKC (24). Firm evidence for PKA-mediated phosphorylation of CFTR as the primary regulator of CFTR activity comes from studies showing that altering or removing sites of in vivo phosphorylation in the R domain reduces, but does not eliminate, PKA stimulation of CFTR in intact cells and excised patches (4, 27, 33). However, the role of PKC in regulating the CFTR channel is less clear.

Addition of phorbol ester potentiated cAMP responses in Xenopus oocytes expressing wild-type CFTR (28) and in HT-29 colonic cells (1), T84 cells (6), C127 cells (7), and pancreatic duct cells (34). In membrane patches excised from cells expressing CFTR, addition of exogenous PKC caused a modest increase in CFTR channel activity and enhanced the rate and magnitude of subsequent PKA stimulation of open probability (29). Nevertheless, the interpretation of effects of phorbol 12-myristate 13-acetate (PMA) is not uniform and, instead, varies from direct PKC regulation of CFTR channel (6, 7, 29) to PMA-mediated increase in cell membrane area (34) and also PKC-mediated de novo insertion of channels into the plasma membrane (1). Trying to pinpoint how PKC regulates CFTR is complicated, however, by the use of PMA, which also influences CFTR expression (30) and degradation (3). Moreover, although PKC is considered to be the major receptor of PMA, its interactions with other enzymes might obscure a specific role for PKC in CFTR function.

Our studies on PKC regulation of a Cl secretory pathway in tracheal epithelial cells (20, 22) led us to test the effects of a potent PKC inhibitor, chelerythrine, on CFTR function. Preliminary data showed that chelerythrine delayed efflux of 36Cl in Calu-3 lung cells stimulated by (-)-epinephrine, a cAMP-generating agent (see Fig. 1), suggesting that constitutive PKC activity in unstimulated cells is necessary for maximal activation of CFTR. Similar findings were reported by Jia et al. (14), from patch-clamp studies of Chinese hamster ovary (CHO) and baby hamster kidney (BHK) cells expressing wild-type CFTR. The question of how PKC regulates CFTR is still unanswered. One step in understanding the regulation of CFTR by PKC is to identify PKC isotype(s) that are required for CFTR function. That is the goal of these studies.

    MATERIALS AND METHODS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Materials. 36Cl (specific activity 260 MBq/g, 7.5 mCi/g) was purchased from ICN Radiochemical (Irvine, CA) and [gamma -32P]ATP (specific activity 111 TBq/mmol, 3,000 Ci/mol) was purchased from Amersham Life Science (Arlington Heights, IL). An enhanced chemiluminescence kit was purchased from Amersham, and the PKC assay system, PKA assay system, and Lipofectin reagent were from GIBCO BRL Life Technologies (Gaithersburg, MD). KN-93 and chelerythrine chloride were purchased from Calbiochem (La Jolla, CA), PMA and forskolin were obtained from Research Biochemicals International (Natick, MA), and 8-(4-chlorophenylthio)adenosine 3',5'-cyclic monophosphate (CPT-cAMP) and (-)-epinephrine were from Sigma (St. Louis, MO). Rabbit polyclonal anti-PKC isotype-specific antibodies were purchased from Santa Cruz Biotechnology (Santa Cruz, CA), and monoclonal mouse anti-human CFTR (epitope 729-736 of the R domain) was purchased from Genzyme (Cambridge, MA). Tissue culture supplies were obtained from GIBCO BRL (Grand Island, NY) and Sigma. All other chemicals were reagent grade.

Cell isolation and culture. The Calu-3 cell line (American Type Culture Collection HTB-55; Ref. 26) was grown submerged in Eagle's medium with Earle's balanced salt solution and 10% fetal bovine serum (FBS) at 37°C in a humidified CO2 incubator. For experiments, cells were seeded onto six-well tissue culture plastic dishes and grown to confluence, typically 7-8 days after subculture. A CHO cell line stably expressing wild-type CFTR (CHO-wtCFTR) was generously given to us by Dr. J. Riordan (Mayo Clinic, Scottsdale, AZ). The CHO-wtCFTR cell line was maintained in culture medium consisting of alpha MEM, 8% FBS, 200 µM methotrexate, and 1% penicillin-streptomycin. CHO cells were grown to confluence in a 5% CO2 incubator at 37°C.

Measurement of CFTR activity as Cl efflux. CFTR activity was assayed by measuring the rate of 36Cl efflux (32). Cell cultures were grown to confluence in six-well tissue culture dishes and preincubated in serum-free medium for 24 h before use. Cells were preincubated for 1 h at 35°C with 3.5 µCi 36Cl in 10 mM HEPES-buffered Ringer (HBR; pH 7.5) solution that contained (in mM) 136.9 NaCl, 5.4 KCl, 0.4 KH2PO4, 0.3 NaHPO4, 4.2 NaHCO3, 1.3 CaCl2, 0.5 MgCl2, 0.4 MgSO4, and 5.6 D-glucose. Medium with radioactive tracer was removed, and cells were washed four times with HBR to remove extracellular 36Cl. After the wash, 0.5-ml aliquots of isotope-free HBR were added and sequentially removed every 60 s for up to 11 min. The first three aliquots were used to establish a stable baseline in efflux buffer only. Agonists were added after the third aliquot was removed. Inhibitors were present in the bathing medium for the last 30 min of the 36Cl loading period and during the efflux period. Radioactive counts remaining in the cells were extracted with 0.1 N NaOH. The fraction of intracellular 36Cl remaining in the cell layer during each time point was calculated from the sample and extract counts. Time-dependent rates of 36Cl efflux were calculated as ln (36Clt = 1/36Clt = 2)/(t1 - t2), where 36Cl is the percent intracellular Cl at time t and t1 and t2 are successive time points.

Oligonucleotide treatment of cells. Phosphodiester oligonucleotides were purchased from GIBCO BRL. Antisense oligonucleotides to PKC isotypes were complementary to the translation initiation region of mRNA specific for the animal species delineated in Table 1. Sense oligonucleotides were used as controls. Oligonucleotides were dissolved in sterile deionized water to a final concentration of 1 mM, aliquoted, and stored at -20°C until ready for use.

                              
View this table:
[in this window]
[in a new window]
 
Table 1.   Oligonucleotide sequences of PKC isotypes

Oligonucleotides were added to inside wells of confluent cell cultures in combination with the cationic lipid N-[1,(2,3-dioleyloxy)propyl]-n,n,n-trimethylammonium chloride and dioleoyl phosphatidylethanolamine lipid (Lipofectin) in serum- and antibiotic-free culture medium, as previously described (20). Oligonucleotide incubation medium was replaced every 12 h for 48 h.

Immunoblot analysis of CFTR and PKC isotypes. Culture medium was replaced with Hanks' balanced salt solution supplemented with 10 mM HEPES (pH 7.5). Cells were treated with vehicle or drugs of interest at 35°C for times indicated. Cell cultures were immediately washed twice with ice-cold PBS and then harvested in 1 ml 100 mM NaCl, 50 mM NaF, 50 mM Tris · HCl (pH 7.55), 1% NP-40, 0.25% sodium deoxycholate, 1 mM EDTA, 1 mM EGTA, 1 mM sodium vanadate, 100 µM leupeptin, 1 µg/ml aprotinin, and 1 mM 4-(2-aminoethyl)benzenesulfonylfluoride hydrochloride. Immunoblot analysis of cell proteins for PKC isotypes was performed as described previously (22). Protein bands immunoreactive to specific antibodies were detected using enhanced chemiluminescence and analyzed by laser densitometry in a Sciscan 5000 (United States Biochemical), using the OS-Scan image analysis system software package (Oberlin Scientific).

To confirm the expression of wtCFTR in transfected cells and CFTR in Calu-3 cells, immunoblot analysis for CFTR was performed on lysates of cells. Lysis buffer was supplemented with 0.1% SDS. Lysates were incubated at 30°C for 30 min in 50 mM Tris (pH 6.8), 100 mM dithiothreitol, 5% glycerol, 4% SDS (wt/vol), and 0.1 beta -mercaptoethanol (vol/vol) and centrifuged at 12,500 g for 2 min to pellet particulate material. Immunoblot analysis was performed on proteins separated on 6% SDS-polyacrylamide gels for CFTR using monoclonal antibody to the R domain of CFTR. CFTR was detected in Calu-3 cells and in CHO-wtCFTR cells (data not shown).

Measurement of PKC and PKA activity. Cell cultures grown on 60-mm tissue culture plastic dishes were treated with vehicle or the drug of interest and then harvested in 0.5 ml of lysis buffer. Lysates were clarified and incubated with antiserum against a specific PKC isotype, as previously described (22). Kinase activity of immune complexes of PKC isotypes was measured using histone III as the substrate for PKC-alpha , -beta II, -delta , and -zeta and a peptide derived from the pseudosubstrate region of PKC-epsilon as the substrate for PKC-epsilon (25). PKA activity of clarified lysates was measured using a commercially available assay system (GIBCO BRL Life Technologies).

Data analysis. Protein levels were determined with a Bradford assay kit (Bio-Rad, Hercules, CA) using BSA as the standard. Data were analyzed by ANOVA followed by Bonferroni multiple comparison tests or by Student's t-tests for unpaired samples. Data are reported as means ± SE for the number of cell monolayers tested (n).

    RESULTS
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Effect of PKC inhibitor on cAMP-stimulated efflux. CFTR function has been assessed in CHO-wtCFTR and Calu-3 cells using iodide efflux and whole cell and patch-clamp recordings of cell-attached and cell-excised patches (13, 29). These methods detected a cAMP-regulated Cl permeability that was indistinguishable from CFTR. For the studies reported here, 36Cl efflux was used as an indicator of CFTR function. Figure 1 illustrates the effect of chelerythrine, a general PKC inhibitor, on CFTR function in CHO-wtCFTR and Calu-3 cells. Addition of the combination of forskolin and CPT-cAMP to CHO-wtCFTR cells rapidly increased the rate of 36Cl efflux, with peak rates at 2 min after addition of stimulatory agents (Fig. 1A). Efflux rates subsequently declined to the steady-state level observed just before addition of stimulatory agents. Calu-3 cells gave a similar response to 3 µM (-)-epinephrine, an endogenous hormone that occupies alpha - and beta -adrenergic receptors and increases cAMP levels (Fig. 1B). Maximal rates of 36Cl efflux occurred 1-2 min after addition of hormone and declined afterward to prestimulatory steady-state levels. Figure 2 summarizes the sensitivity of CFTR activity at peak rates to cAMP-generating agents. The combination of forskolin and CPT-cAMP significantly increased the rate of efflux in CHO-wtCFTR to 0.78 ± 0.11 min-1 (n = 9; Fig. 2A) and in Calu-3 cells to 0.80 ± 0.16 min-1 (n = 5; Fig. 2B). As seen in Fig. 2B, forskolin and CPT-cAMP mimicked the effect of (-)-epinephrine in Calu-3 cells. A signaling mechanism for the effects of forskolin plus CPT-cAMP focuses on the elevation of cAMP levels by bypassing membrane receptors, which leads to activation of PKA and subsequent phosphorylation of CFTR and increased channel activity. However, the data of Fig. 1 suggest a role for PKC in cAMP-dependent activation of CFTR. Figure 1 shows that pretreatment with 10 µM chelerythrine, a general PKC inhibitor, abolished the stimulatory effects of forskolin and CPT-cAMP on CHO-wtCFTR cells and of (-)-epinephrine on Calu-3 cells, suggesting that PKC activity in unstimulated cells regulates CFTR function. As seen in Fig. 2A, chelerythrine significantly reduced peak rates elicited with cAMP-generating agents and, more importantly, with the phorbol ester PMA, an activator of PKC, in CHO-wtCFTR cells. The sensitivity of PMA-induced CFTR activity to chelerythrine indicates that inhibition of PKC blocks PKC-induced CFTR activity.


View larger version (16K):
[in this window]
[in a new window]
 


View larger version (15K):
[in this window]
[in a new window]
 
Fig. 1.   Detection of cystic fibrosis transmembrane conductance regulator (CFTR) activity in CHO-wtCFTR (A) and Calu-3 (B) cell lines. Rate of 36Cl efflux (r) is plotted as function of time. Chelerythrine chloride, at final concentration of 10 µM, was added to cells 30 min before start of radioisotopic efflux. Stimulatory agents were added immediately after sampling medium at 3 min and were present during remainder of efflux period. A: , vehicle (n = 10); bullet , 10 µM forskolin and 50 µM CPT-cAMP (n = 9); open circle , forskolin and CPT-cAMP + chelerythrine (n = 5). B: , vehicle (n = 25); , chelerythrine (n = 5); bullet , 3 µM (-)-epinephrine (n = 6); open circle , (-)-epinephrine + chelerythrine (n = 4). Error bars are SE. * P < 0.001 compared with vehicle; # P < 0.02, ## P < 0.005 compared with cells treated with cAMP-generating agent.


View larger version (14K):
[in this window]
[in a new window]
 


View larger version (17K):
[in this window]
[in a new window]
 
Fig. 2.   Sensitivity of 36Cl efflux from CHO-wtCFTR (A) and Calu-3 (B) cells to stimulatory agents. Data are taken from Fig. 1 at a time point 2 min after addition of vehicle or stimulatory agent. Percentages indicate component of radioisotopic efflux that was sensitive to 10 µM chelerythrine. Final concentrations were 1 µM phorbol 12-myristate 13-acetate (PMA), 10 µM forskolin (FSK), 50 µM CPT-cAMP, and 3 µM (-)-epinephrine (EPI). Error bars are SE for 4-10 replicate experiments for each condition. * P < 0.03, ** P < 0.001 compared with cells treated with vehicle; # P < 0.02, compared with cells treated with stimulatory agent alone.

In Calu-3 cells, responses to forskolin plus CPT-cAMP were also markedly sensitive to preincubation with chelerythrine (Fig. 2B). A comparison of the two cell lines shows that chelerythrine reduced peak Cl efflux to rates observed in cells treated only with vehicle. Inhibition by chelerythrine affected 69.4% of peak Cl efflux in CHO-wtCFTR cells and 79.2% of peak Cl efflux in Calu-3 cells. The data of Fig. 2 also show that the two cell lines differed in the rates of baseline Cl efflux in cells exposed only to vehicle. CHO-wtCFTR cells consistently displayed a 2.0-fold higher rate compared with Calu-3 cells (P < 0.001). This might indicate that, in cells grown in this laboratory, there is constitutive CFTR activity in unstimulated, stably transfected CHO-wtCFTR cells. (-)-Epinephrine induced a smaller increase in Cl efflux than did forskolin plus CPT-cAMP. One possible explanation for this response is activation of intracellular signaling pathways in addition to cAMP-generating pathways through alpha 1-adrenergic receptors (19), leading to increased activity of PKC-delta and -zeta with subsequent modulation of cAMP-increased CFTR function in addition to basolateral Na-K-2Cl cotransport (22).

Effect of antisense oligonucleotides on cAMP-dependent CFTR activation. The approach of antisense technology to reduce PKC isotype mass and activity was used by this laboratory to identify a critical role for PKC-delta in the regulation of Na-K-2Cl cotransport in human tracheal epithelial cells (20) and in CF/T43 cells (21). Calu-3 cells were used in the next series of experiments, in which an antisense approach was used to mimic the effect of inhibition of PKC by chelerythrine on CFTR function. First, PKC isotypes expressed by Calu-3 cells were identified by immunoblot analysis. Calu-3 cells were immunoreactive with polyclonal antibodies to PKC-alpha , -beta II, -delta , -epsilon , and -zeta (Fig. 3A). PKC-beta I, -gamma , and -eta were not detected. Proteins immunoreactive to PKC isotypes corresponded closely in apparent molecular weight to recombinant PKC isotypes and to calculated PKC isotype molecular weights. Immunoreactive protein bands to PKC isotypes that were found in Calu-3 cells were also found in normal human tracheal epithelial cells (22) and in CF/T43 cells (21). The relative distribution of PKC isotypes to cytosol and a particulate fraction in Calu-3 cells (Fig. 3A) shared similarities with normal human tracheal epithelial cells (22) and with CF/T43 cells (21), with PKC-alpha and -zeta localized predominantly in cytosol and PKC-beta II distributed approximately evenly between cytosol and a particulate fraction. PKC-delta and -epsilon were distributed predominantly to cytosol.


View larger version (55K):
[in this window]
[in a new window]
 


View larger version (51K):
[in this window]
[in a new window]
 
Fig. 3.   Immunoblot analysis of protein kinase C (PKC) isotypes in Calu-3 cells. A: Calu-3 cells were lysed in hypotonic buffer and fractionated into cytosol (C) and a particulate (P) fraction, as previously described (22); 20 µg of homogenate (T), cytosol, and particulate fraction were subjected to electrophoresis on 8% SDS-PAGE gels. Protein bands were transferred to Immobilon papers and probed with polyclonal antibody to PKC isotypes, as previously described (22). Laser densitometry of bands detected in cytosol and particulate fractions was performed to obtain portion of total PKC isotype mass in cytosol and particulate fraction that was detected in particulate fraction (%P). B: cells were untreated (UT) or incubated with Lipofectin + antisense oligonucleotide (AS) to PKC-delta , -epsilon , or -zeta as described in MATERIALS AND METHODS; 20 µg of whole cell lysate were electrophoresed and analyzed as described for A.

Next, cells were cultured in the presence of antisense oligonucleotide to PKC-delta , -epsilon , or -zeta for 48 h. Optimal concentrations of oligonucleotide and Lipofectin were determined from dose-response curves. Antisense oligonucleotide to PKC-delta or -zeta did not alter (-)-epinephrine-stimulated 36Cl efflux (Table 2), even though PKC-delta and -zeta mass were reduced by 73.7 ± 1.5 (n = 3) and 86.1 ± 0.3% (n = 3), respectively, without affecting PKC-alpha , -beta II, or -epsilon (Fig. 3B). Antisense oligonucleotide to PKC-epsilon , on the other hand, abolished stimulation of CFTR by (-)-epinephrine (Table 2 and Fig. 4). Baseline CFTR function was not significantly affected by treatment with Lipofectin or oligonucleotides (Fig. 4B). Cells pretreated with sense oligonucleotide to PKC-epsilon retained the stimulatory response to (-)-epinephrine and a chelerythrine-sensitive component of Cl flux similar to untreated and Lipofectin-treated cells (Fig. 4B). These results indicate that PKC-epsilon regulates CFTR function.

                              
View this table:
[in this window]
[in a new window]
 
Table 2.   Effect of antisense oligonucleotides to PKC isotypes on CFTR function


View larger version (22K):
[in this window]
[in a new window]
 


View larger version (27K):
[in this window]
[in a new window]
 
Fig. 4.   Inhibition of cAMP-stimulated CFTR activity by antisense oligonucleotide to PKC-epsilon . A: rate of 36Cl efflux is plotted as function of time. Cell monolayers were incubated without lipids or nucleotide (UT; n = 25), with 1 µg/ml Lipofectin (LPF; n = 12), or with Lipofectin + antisense oligonucleotide to PKC-epsilon (AS-PKC-epsilon ; n = 12). Cells were stimulated with vehicle or with 3 µM (-)-epinephrine. Error bars are SE. B: plot of peak rates of 36Cl efflux. Cell monolayers were incubated with 1 µM antisense oligonucleotide to PKC-epsilon (AS-epsilon; n = 8) or 1 µM sense oligonucleotide to PKC-epsilon (SS-epsilon; n = 8) for 48 h. Cells were treated with vehicle (open bar), 10 µM chelerythrine (crosshatched bar), 3 µM (-)-epinephrine (hatched bar), or epinephrine + chelerythrine (solid bar). Percentages refer to component of rate of efflux attributed to PKC-sensitive mechanism. Error bars are SE. * P < 0.05, ** P < 0.001 compared with cells treated with vehicle; # P < 0.03, ## P < 0.0001 compared with cells treated with (-)-epinephrine alone.

Effect of antisense oligonucleotide on PKC-epsilon expression and activity. Antisense oligonucleotide to PKC-epsilon could block activation of CFTR by cAMP by diminishing PKC-epsilon activity in unstimulated cells, decreasing the amount of PKC-epsilon , or both. The first possibility was investigated by measuring kinase activity of immune complexes of PKC isotypes in untreated cells or in cells preincubated for 24 h with 1 µg/ml Lipofectin or with Lipofectin plus antisense oligonucleotide to PKC-epsilon . Incubation of Calu-3 cells with antisense oligonucleotide reduced total PKC-epsilon activity by 52.1% (Table 3). Moreover, PKC-epsilon activity per unit protein also significantly decreased, indicating a loss of PKC-epsilon activity. Treatment of cells with Lipofectin did not significantly alter baseline PKC-epsilon activity (Table 3). PKC-epsilon expression was next evaluated in cells treated with antisense oligonucleotide to PKC-epsilon and, as a control, in untransfected cells (Fig. 3B). Antisense oligonucleotide reduced PKC-epsilon by 76.1 ± 4.5% (n = 6) and did not affect PKC-alpha , -beta II, -delta , or -zeta mass (Fig. 3B). These results indicate that antisense oligonucleotide to PKC-epsilon blocks CFTR function by decreasing PKC-epsilon mass and activity in Calu-3 cells.

                              
View this table:
[in this window]
[in a new window]
 
Table 3.   Effect of antisense oligonucleotides on activity of PKC-epsilon in Calu-3 cells

Effect of antisense oligonucleotide on PKA activity. Regulation of CFTR function by PKC-epsilon could be due to modulation of PKA to alter PKA-dependent phosphorylation of CFTR. To test this possibility, PKA activity was quantitated in untransfected cells, Lipofectin-treated cells, and cells transfected with antisense oligonucleotide to PKC-epsilon . As seen in Table 4, antisense oligonucleotide to PKC-epsilon did not affect (-)-epinephrine-stimulated PKA activity, indicating the independence of PKA from PKC-epsilon . Therefore, PKC-epsilon regulates CFTR function at a different site.

                              
View this table:
[in this window]
[in a new window]
 
Table 4.   (-)-Epinephrine-stimulated PKA activity is unaffected by antisense oligonucleotide to PKC-epsilon

    DISCUSSION
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

Regulation of CFTR Cl channel by PKC has been inferred from studies of CFTR expressed in a variety of epithelial cell lines and in heterologous expression systems (1, 6, 7, 14, 28, 34), yet few studies provide detailed information about PKC regulation of CFTR. The current study provides the first report of regulation of CFTR channel function by a Ca-independent PKC isotype, PKC-epsilon . The cAMP-generating agent forskolin in combination with CPT-cAMP increased CFTR Cl channel activity, measured as 36Cl efflux, from 0.23 ± 0.02 min-1 (n = 10) to 0.78 ± 0.11 min-1 (n = 9) in CHO-wtCFTR cells and from 0.12 ± 0.01 min-1 (n = 25) to 0.80 ± 0.16 min-1 (n = 5) in Calu-3 cells. Pretreatment of both cell lines with the general PKC inhibitor chelerythrine blocked cAMP-induced CFTR Cl channel activity (Figs. 1 and 2), suggesting that baseline, constitutive PKC activity is necessary for acute stimulation of CFTR. Jia et al. (14) came to a similar conclusion from a study of two mammalian expression systems, CHO-wtCFTR and BHK-wtCFTR, using an electrophysiological approach. The studies reported here also show that elevated CFTR function induced by PMA in CHO-wtCFTR cells (Fig. 2A) was blocked by pretreatment with chelerythrine, indicating that chelerythrine blocks the effects of PMA by preventing an increase in PKC activity.

Previous studies by this laboratory on basolateral Na-K-2Cl cotransport showed that, in the absence of maximal Cl secretion, activation of cotransport by alpha 1-adrenergic agents requires a PKC signaling mechanism and that increased PKC-delta activity in a cytosolic fraction is necessary for activation (20). The identity of a PKC isotype necessary for cAMP-dependent CFTR Cl channel activity was investigated first by establishing which PKC isotypes were expressed in Calu-3 cells and then by mimicking the effects of inhibition of PKC enzyme activity by individually downregulating PKC isotypes. Calu-3 cells express five PKC isotypes representative of three major types of PKC isotypes (Fig. 3). Conventional PKC isotypes alpha  and beta II, novel PKC isotypes delta  and epsilon , and atypical PKC isotype zeta  were identified by immunoblot analysis. The same PKC isotypes were also found in human tracheal epithelial cell cultures and in CF/T43 cells, indicating that this panel of PKC isotypes is a signature of tracheal epithelial cells and cell lines derived from primary tracheal epithelial cell cultures (20, 21).

Downregulation of PKC isotypes was accomplished using an antisense approach. Using this approach, this laboratory obtained convincing evidence for a role for PKC-delta in the activation of Na-K-2Cl cotransport. A major reason for using this approach is that long-term treatment with the phorbol ester PMA for 18 h did not deplete PKC isotype activity or abundance in CF/T43 cells (21) or in primary cultures of human tracheal epithelial cells (22). Treatment of Calu-3 cells with antisense oligonucleotide to PKC-delta or -zeta for 48 h reduced PKC-delta by 73.7% and PKC-zeta by 81.1%, respectively, but failed to prevent cAMP-dependent CFTR Cl channel function (Table 2) and did not affect Cl loading. Previous studies established that antisense oligonucleotide to PKC-delta prevents alpha 1-adrenergic activation of Na-K-2Cl cotransport but not baseline cotransport activity in unstimulated cells. Thus there may be sufficient cotransport activity in cells treated with antisense oligonucleotide to PKC-delta to support Cl loading. Alternatively, activity of other Cl transport pathways (e.g., Cl/HCO3 exchange) might mediate Cl loading. A major finding of this study is that antisense oligonucleotide to PKC-epsilon potently blocked cAMP-dependent activation of CFTR Cl channel function (Table 3 and Fig. 4). Antisense oligonucleotide to PKC-epsilon also reduced PKC-epsilon by 76.1%, a finding consistent with the half-life of ~24 h for PKC in vitro, and significantly reduced baseline activity of PKC-epsilon (Table 3). The latter finding provides evidence for constitutive PKC-epsilon activity that was also suggested by Jia et al. (14) to explain their results.

A PKC-epsilon signaling mechanism involved in regulation of CFTR function was next investigated by asking whether PKC-epsilon modulated activity of PKA, a protein kinase that is specifically activated by cAMP and is necessary for increased CFTR Cl channel activity. Antisense oligonucleotide to PKC-epsilon did not affect a (-)-epinephrine-mediated increase in PKA activity (Table 4) despite blocking (-)-epinephrine-induced CFTR Cl channel activity (Fig. 4). Hence, PKC-epsilon regulates CFTR Cl channel function at a site other than PKA. The phorbol ester PMA stimulates CFTR Cl channel function, apparently by increasing PKC phosphorylation of CFTR; however, more definitive studies are needed to determine whether phosphorylation of CFTR by PKC-epsilon in unstimulated cells is necessary to achieve maximal CFTR channel activity when cAMP levels are elevated.

Downregulation of PKC-epsilon by antisense oligonucleotides or by long-term phorbol ester treatment has implicated PKC-epsilon in a number of cellular functions, including regulation of electrolyte and nonelectrolyte transporters. In a human liver epithelial BC1 cell line, long-term PMA treatment reduced CFTR mRNA, with a concomitant inhibition of CFTR Cl channel activity, and induced a cytosol-to-membrane translocation of PKC-alpha and -epsilon (16), indicating a role for these PKC isotypes in previously observed PMA-sensitive CFTR expression (30). PKC-epsilon was also found to be necessary for inhibition of vasopressin-stimulated Na transport in rabbit cortical collecting duct cells, suggesting cAMP-mediated activation of PKC-epsilon (8), and for amino acid transport in cultured human fibroblasts (11). In rat cardiac muscle, PKC-epsilon translocation to cross-striated structures after stimulation with PMA or with alpha 1-adrenergic agonists (10) can be blocked with chelerythrine or the V1 fragment of PKC-epsilon (10), resulting in loss of contractility (9, 15) and protection from ischemic injury (12). PKC isotypes, either alone or in combination with other PKC isotypes, play major roles in cross talk among signal transduction pathways, as demonstrated in recent studies. For example, mechanosensitive signal transduction in endothelial cells involves PKC-epsilon and extracellular signal-regulated kinase-1/2 (31), and tumor necrosis factor-alpha -regulated insulin signaling in HEK-293 cells involves PKC-epsilon (17). However, selective activation of PKC-alpha or -epsilon in rat adrenal glomerulosa cells is stimulus dependent (23), and the level of expression of human endothelial nitric oxide synthase increases with PMA-stimulated PKC-alpha and -epsilon (18).

Identification of a Ca-independent PKC isotype in the regulation of CFTR agrees with the phosphorylation studies of Picciotto et al. (24), who report that PKC phosphorylated CFTR in a Ca-independent manner. Phosphopeptide mapping localized PKC phosphorylation sites at serines 686 and 790 of the R domain; however, in vivo PKC phosphorylation using PMA as a PKC activator revealed additional sites outside the R domain. The role of PKC phosphorylation sites is still not clear, although recent studies by Wilkinson et al. (33) suggest an interaction between phosphorylated amino acid residues to explain roles for inhibitory and stimulatory phosphorylation sites (16).

The finding that PKC-epsilon regulates CFTR function is novel and clearly differentiates PKC-epsilon regulation of CFTR from PKC-delta modulation of Na-K-2Cl cotransport. Although both PKC isotypes are diacylglycerol dependent and Ca2+ independent, cotransport activation requires increased PKC-delta activity as opposed to an apparently constitutive PKC-epsilon activity linked to CFTR. These results indicate specific roles for selective PKC isotypes in airway epithelial cells and, furthermore, suggest that a critical component of a PKC signaling mechanism is the targeting of a PKC isotype to its substrate protein, which can be localized to apical or basolateral membranes.

    ACKNOWLEDGEMENTS

We acknowledge the excellent technical assistance of Qiuping Shu and Peter Wung.

    FOOTNOTES

This research was supported by National Heart, Lung, and Blood Institute Grant HL-50160 and by the Cystic Fibrosis Foundation.

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

Address for reprint requests: C. M. Liedtke, Pediatric Pulmonology, Case Western Reserve University, BRB, Room 824, 2109 Adelbert Rd., Cleveland, OH 44106-4948.

Received 19 May 1998; accepted in final form 12 August 1998.

    REFERENCES
Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References

1.   Bajnath, R. B., J. A. Groot, H. R. DeJonge, M. Kansen, and J. Bijman. Synergistic activation of non-rectifying small-conductance chloride channels by forskolin and phorbol esters in cell-attached patches of the human colon carcinoma cell line HT-29cl.19A. Pflügers Arch. 425: 100-106, 1993[Medline].

2.   Basta, P., M. B. Strickland, W. Holmes, C. R. Loomis, L. M. Ballas, and D. J. Burns. Sequence and expression of human protein kinase C-epsilon. Biochim. Biophys. Acta 1132: 154-160, 1992[Medline].

3.   Breuer, W., H. Glickstein, N. Kartner, J. R. Riordan, D. A. Ausiello, and I. Z. Cabantchik. Protein kinase C mediates down-regulation of cystic fibrosis transmembrane regulator levels in epithelial cells. J. Biol. Chem. 268: 13935-13939, 1993[Abstract/Free Full Text].

4.   Chang, X.-B., J. A. Tabcharani, Y.-X. Hou, T. J. Jensen, N. Kartner, N. Alon, J. W. Hanrahan, and J. R. Riordan. Protein kinase A (PKA) still activates CFTR chloride channel after mutagenesis of all 10 PKA consensus phosphorylation sites. J. Biol. Chem. 268: 11304-11311, 1993[Abstract/Free Full Text].

5.   Cohn, J. A., A. C. Nairn, C. R. Marino, O. Melhus, and J. Kole. Characterization of the cystic fibrosis transmembrane conductance regulator in a colonocyte cell line. Proc. Natl. Acad. Sci. USA 89: 2340-2344, 1992[Abstract].

6.   Dechecchi, M. C., R. Rolfini, A. Tamanini, C. Gamberi, G. Berton, and G. Cabrini. Effect of modulation of protein kinase C on the cAMP-dependent chloride conductance in T84 cells. FEBS Lett. 311: 25-28, 1992[Medline].

7.   Dechecchi, M. C., A. Tamanini, G. Berton, and G. Cabrini. Protein kinase C activates chloride conductance in C127 cells stably expressing the cystic fibrosis gene. J. Biol. Chem. 268: 11321-11325, 1993[Abstract/Free Full Text].

8.   DeCoy, D. L., J. R. Snapper, and M. D. Breyer. Antisense DNA down-regulates protein kinase C-epsilon and enhances vasopressin-stimulated Na+ absorption in rabbit cortical collecting duct. J. Clin. Invest. 95: 2749-2756, 1995[Medline].

9.   Deng, X.-F., S. Mulay, and D. R. Varma. Role of Ca2+-independent PKC in alpha 1-adrenoreceptor-mediated inotropic responses of neonatal rat hearts. Am. J. Physiol. 273 (Heart Circ. Physiol. 42): H1113-H1118, 1997[Abstract/Free Full Text].

10.   Disatnik, M.-H., G. Buraggi, and D. Mochly-Rosen. Localization of protein kinase C isozymes in cardiac myocytes. Exp. Cell Res. 210: 287-297, 1994[Medline].

11.   Franchi-Gazzola, R., R. Visigalli, O. Bussolati, and G. C. Gazzola. Involvement of protein kinase Cepsilon in the stimulation of anionic amino acid transport in cultured human fibroblasts. J. Biol. Chem. 271: 26124-26130, 1996[Abstract/Free Full Text].

12.   Gray, M. O., J. S. Karliner, and D. Mochly-Rosen. A selective epsilon -protein kinase C antagonist inhibits protection of cardiac myocytes from hypoxia-induced cell death. J. Biol. Chem. 272: 30945-30951, 1997[Abstract/Free Full Text].

13.   Haws, C., W. E. Finkbeiner, J. H. Widdicombe, and J. J. Wine. CFTR in Calu-3 human airway cells: channel properties and role in cAMP-activated Cl- conductance. Am. J. Physiol. 266 (Lung Cell. Mol. Physiol. 10): L502-L512, 1994[Abstract/Free Full Text].

14.   Jia, Y., C. J. Mathews, and J. W. Hanrahan. Phosphorylation by protein kinase C is required for acute activation of cystic fibrosis transmembrane conductance regulator by protein kinase A. J. Biol. Chem. 272: 4978-4984, 1997[Abstract/Free Full Text].

15.   Johnson, J. A., M. O. Gray, C.-H. Chen, and D. Mochly-Rosen. A protein kinase C translocation inhibitor as an isozyme-selective antagonist of cardiac function. J. Biol. Chem. 271: 24962-24966, 1966[Abstract/Free Full Text].

16.   Kang-Park, S., N. Dray-Charier, A. Munier, C. Brahimi-Horn, D. Veissiere, J. Picard, J. Capeau, G. Cherqui, and O. Lascols. Role for PKC alpha and PKC epsilon in down-regulation of CFTR mRNA in a human epithelial liver cell line. J. Hepatol. 28: 250-262, 1998[Medline].

17.   Kellerer, M., J. Mushack, H. Mischak, and H. U. Haring. Protein kinase C (PKC) epsilon enhances the inhibitory effect of TNF alpha on insulin signaling in HEK293 cells. FEBS Lett. 418: 119-122, 1997[Medline].

18.   Li, H., S. A. Oehrlein, T. Wallerath, I. Ihrig-Biedert, P. Wohlfart, T. Ulshofer, T. Jessen, T. Herget, U. Forstermann, and H. Kleinert. Activation of protein kinase C alpha and/or epsilon enhances transcription of the human endothelial nitric oxide synthase gene. Mol. Pharmacol. 53: 630-637, 1998[Abstract/Free Full Text].

19.   Liedtke, C. M. The role of protein kinase C in alpha -adrenergic regulation of NaCl(K) cotransport in human airway epithelial cells. Am. J. Physiol. 268 (Lung Cell. Mol. Physiol. 12): L414-L423, 1995[Abstract/Free Full Text].

20.   Liedtke, C. M., and T. Cole. Antisense oligodeoxynucleotide to PKC-delta blocks alpha 1-adrenergic activation of Na-K-2Cl cotransport. Am. J. Physiol. 273 (Cell Physiol. 42): C1632-C1640, 1997[Abstract/Free Full Text].

21.   Liedtke, C. M., and T. S. Cole. Antisense oligodeoxynucleotide to PKCdelta downregulates PKCdelta mass and activity and suppresses alpha 1-adrenergic activation of Na-Cl-K cotransport in human tracheal epithelial cells. Pediatr. Pulmonol. Suppl. 14: 243, 1997.

22.   Liedtke, C. M., T. Cole, and M. Ikebe. Differential activation of PKC-delta and -zeta by alpha 1-adrenergic stimulation in human airway epithelial cells. Am. J. Physiol. 273 (Cell Physiol. 42): C937-C943, 1997[Abstract/Free Full Text].

23.   Natarajan, R., L. Lanting, L. Xu, and J. Nadler. Role of specific isoforms of protein kinase C in angiotensin II and lipoxygenase action in rat adrenal glomerulosa cells. Mol. Cell. Endocrinol. 101: 59-66, 1994[Medline].

24.   Picciotto, M. R., J. A. Cohn, G. Bertuzzi, P. Greengard, and A. C. Nairn. Phosphorylation of the cystic fibrosis transmembrane conductance regulator. J. Biol. Chem. 267: 12742-12752, 1992[Abstract/Free Full Text].

25.   Schaap, D., P. J. Parker, A. Bristol, R. Kriz, and J. Knopf. Unique substrate specificity and regulatory properties of PKC-epsilon : a rationale for diversity. FEBS Lett. 243: 351-357, 1989[Medline].

26.   Shen, B.-Q., W. E. Finkbeiner, J. J. Wine, R. J. Mrsny, and J. H. Widdicombe. Calu-3: a human airway epithelial cell line that shows cAMP-dependent Cl- secretion. Am. J. Physiol. 266 (Lung Cell. Mol. Physiol. 10): L493-L501, 1994[Abstract/Free Full Text].

27.   Siebert, F. S., J. A. Tabcharani, X.-B. Chang, A. M. Dulhanty, C. Mathews, J. W. Hanrahan, and J. R. Riordan. cAMP-dependent protein kinase-mediated phosphorylation of cystic fibrosis transmembrane conductance regulator residue Ser-753 and its role in channel activation. J. Biol. Chem. 270: 2158-2163, 1995[Abstract/Free Full Text].

28.   Sullivan, S. K., K. Swamy, and M. Field. cAMP-activated Cl conductance is expressed in Xenopus oocytes by injection of shark rectal gland mRNA. Am. J. Physiol. 260 (Cell Physiol. 29): C664-C669, 1991[Abstract/Free Full Text].

29.   Tabcharani, J. A., X.-B. Chang, J. R. Riordan, and J. W. Hanrahan. Phosphorylation-regulated Cl- channel in Cho cells stably expressing the cystic fibrosis gene. Nature 352: 628-631, 1991[Medline].

30.   Trapnell, B. C., P. L. Zeitlin, C.-S. Chu, K. Yoshimura, H. Nakamura, W. B. Guggino, J. Bargon, T. C. Banks, W. Dalemans, A. Pavirani, J.-P. Lecocq, and R. G. Crystal. Down-regulation of cystic fibrosis gene mRNA transcript levels and induction of the cystic fibrosis chloride secretory phenotype in epithelial cells by phorbol ester. J. Biol. Chem. 266: 10319-10323, 1991[Abstract/Free Full Text].

31.   Traub, O., B. P. Monia, N. M. Dean, and B. C. Berk. PKC-epsilon is required for mechano-sensitive activation of ERK1/2 in endothelial cells. J. Biol. Chem. 272: 31251-31257, 1997[Abstract/Free Full Text].

32.   Vanglarik, C. J., R. J. Bridges, and R. A. Frizzell. A simple assay for agonist-regulated Cl and K conductances in salt-secreting cells. Am. J. Physiol. 259 (Cell Physiol. 28): C358-C364, 1990[Abstract/Free Full Text].

33.   Wilkinson, D. J., T. V. Strong, M. K. Mansoura, D. L. Wood, S. S. Smith, F. S. Collins, and D. C. Dawson. CFTR activation: additive effects of stimulatory and inhibitory phosphorylation sites in the R domain. Am. J. Physiol. 273 (Lung Cell. Mol. Physiol. 17): L127-L133, 1997[Abstract/Free Full Text].

34.   Winpenny, J. P., H. L. McAlroy, M. A. Gray, and B. E. Argent. Protein kinase C regulates the magnitude and stability of CFTR currents in pancreatic duct cells. Am. J. Physiol. 268 (Cell Physiol. 37): C823-C828, 1995[Abstract/Free Full Text].


Am J Physiol Cell Physiol 275(5):C1357-C1364
0002-9513/98 $5.00 Copyright © 1998 the American Physiological Society