SUMMARY. Analysis of vectorial ion transport and protein trafficking in transformed cystic fibrosis (CF) epithelial cells has been limited because the cells tend to ...
In VitroCell.Dev.Biol.--Animal31:617-624,Sept 1995 © 1995Societyfor In VitroBiology 1071-2690/95 $05.00+ 0.00
EPITHELIAL
CELL SPECIFIC PROPERTIES AND GENETIC COMPLEMENTATION A AF508 CYSTIC FIBROSIS NASAL POLYP CELL LINE
IN
K. KUNZELMANN,D. C. LEI, K. ENG, L. C. ESCOBAR, T. KOSLOWSKY,ANDD. C. GRUENERT~ Cardiovascular Research Institute and Gene Therapy Core Center (K. K., D. C. L., K. E., L. C. E., D. C. G.), Department of Laboratory Medicine (D. C. G.), University of California at San Francisco, San Francisco, California 94143; and Institut ftir Physiologie, Albert-Ludwigs Universitat, Freiburg, Germany (K. K.).
(Received 24 January 1995; accepted 22 March 1995)
SUMMARY Analysis of vectorial ion transport and protein trafficking in transformed cystic fibrosis (CF) epithelial cells has been limited because the cells tend to lose their tight junctions with multiple subcultures. To elucidate ion transport and protein trafficking in CF epithelial cells, a polar cell line with apical and basolateral compartments will facilitate analysis of the efficacy of different gene therapy strategies in a "tight epithelium" in vitro. This study investigates the genotypic and phenotypic properties of a CF nasal polyp epithelial, AF508 homozygote, cell line that has tight junctions pre-crisis. The cells (ECFNPE14o-) were transformed with an origin-of-replication defective SV40 plasmid. They develop transepithelial resistance in Ussing chambers and are defective in cAMP-dependent CI- transport as measured by efflux of radioactive CI-, short circuit current (I,c), or whole-cell patch clamp. Stimulation of the cells by bradykinin, histamine, or ATP seems to activate both K +- and Ca+2-dependent C1- transport. Measurement of ~6C1- efflux following stimulation with A23187 and ionomycin indicate a Ca+2-dependent CI- transport. Volume regulatory capacity of the cells is indicated by cell swelling conductance. Expression of the CF transmembrane conductance regulator mRNA was indicated by RT-PCR amplification. When cells are grown at 26 ° C for 48 h there is no indication of cAMP-dependent C1- as has been previously indicated in heterologous expression systems. Antibodies specific for secretory cell antigens indicate the presence of antigens found in goblet, serous, and mucous cells; in goblet and serous cells; or in goblet and mucous cells; but not antigens found exclusively in mucous or serous cells. Gene complementation studies with an episomal vector containing wild-type CF transmembrane conductance regulator cDNA showed correction of the cAMP-dependent C1- transport defect. This cell line contributes unique phenotypic features to the store of transformed CF epithelial cells already available. Key words: CF airway epithelial cells; AF508 CFFR; secretory cell; transformation; ion transport; gene therapy.
INTRODUCTION
systems or in non-airway epithelial cells, it is important that these findings be investigated in human airway epithelial cells. Through the development of transformed CF airway epithelial cell lines it has been possible to begin to address some of these questions in a systematic fashion. Numerous human airway epithelial cell lines (both non-CF and CF) have now been developed in our laboratory (4--6,13,14,22) and those of others (2,17,18,31,33). However, very few of these cell lines maintain tight junctions and cell polarity post-crisis or over multiple generations. At present, there are two normal cell lines transformed in vitro that maintain their ability to form tight monolayers and maintain vectorial ion transport over greater than 30 subcultures (5,6,16). Unfortunately, there are no CF airway epithelial cell lines that retain these properties. Because cell polarity and vectorial ion transport may be an important element in expanding our understanding of the effects of genotype on CFTR function as well as the pathology of CF, we have utilized a pre-crisis transformed CF cell line with tight junctions to investigate certain phenotypic properties that might be associated with cell polarity and a more differentiated epithelium. An immortalized celt line (ECFNPE-14o-) was established from a primary culture of CF nasal polyp epithelial cells by transfection
Cystic fibrosis (CF) is the most common lethal genetic disease in the Caucasian population affecting about 1 in 2500 individuals in the population (1,27). This disease impairs the electrolyte transport properties of epithelial cells in the airways, sweat glands, pancreas, and other organs (1). Isolation of the gene responsible for CF, the cystic fibrosis transmembrane conductance regulator (CFTR), was an important step in defining the underlying mechanisms controlling the relationship between CFTR function and genotype. The most common mutation associated with CF, a three base pair deletion that results in a phenylalanine deletion at codon 508 (AF508), is found in ~70% of all CF chromosomes (19,20). Due to its prevalence, the impact of this mutation on CFTR functional is of particular interest. Recent studies have indicated that AF508 CFTR protein does not reach the plasma membrane (3,25,30), but that this transport defect can be overcome to some extent by lowering the cellular temperature (7,25). Because these studies were carried out in heterologous cell To whom correspondence should be addressed at SU203, Box 0911, Cardiovascular Research Institute, University of California, San Francisco, California 94143-0911. 617
618
KUNZELMANNET AL.
with an origin-of-replication defective simian virus 40 (SV40) plasmid (4-6,12-14). Analysis indicates that the cells are homozygous for the AF508 mutation. This cell line is defective in cAMP-dependent C1- transport, but has intact Ca+2-dependent C1- transport as measured by efflux of 36C1 . Furthermore, agents that function through cell surface receptors that activate this Ca+Z-dependent C1 transport pathway are effective in simultaneously stimulating K + transport as has been indicated in previous studies.(21). They also develop transepithelial resistance in Ussing chambers. Characterization of the cells with antibodies specific for secretory cell antigens indicates that ceils express antigens suggestive of goblet cell origin. Gene therapy studies using an episomal vector containing wild-type (wt) CFTR cDNA showed correction of the cAMP-dependent C1transport defect.
MATERIALSAND METHODS Cells and cells transformation. Nasal polyp epithelial cells from a patient with CF were isolated and grown in modified serum-free LHC-9 medium (MLHC-8e) on tissue culture plastic that had been precoated with fibronectin/ vitrogen/bovine serum albumin (FN/V/BSA). Cells were transformed as described previously with the linearized pSVori-plasmid containing a replication-deficient SV40 genome (4-6,12-14,22). Transfected cultures were grown
in MLHC8e medium at 37° C under 5% CO2 in air until cells with altered growth characteristics appeared. Transformants were isolated by trypsinization and expanded for further study. All colonies on the petri dishes were pooled and designated ZCFNPE14o-. After six subcultures the cells were transferred to Eagle's minimal essential medium (MEM) supplemented with 10% fetal bovine serum (FBS) and antibiotics. lmmunocytochemical staining. Cells were grown to confluence on coated well slides. After washing, fixing, and drying the slides were rehydrated and stained for immunnfluorescence. Primary antibodies used were against the SV40 large tumor antigen (Oncogene Science, Uniondale, NY), the AE1/AE3 anticytokeratin antibody (Boehringer Mannheim, Indianapolis, IN), the E9 monoclonal antibody against the junctional complex adhesion protein cellCAM120/80 (29). For light microscopic immunocytochemistry, slides were fixed with 4% paraformaldehyde. Staining was by a modification of a biotin/avidin procedure. Primary antibodies used in these studies included monoclonal antibodies that recognize antigens expressed by goblet, serous, and mucous cells (A1D3), goblet and mucous cells (A1Ell, B6G6, B6E8), or goblet and serous cells (B8C3) and serous cells alone (A2E7, A3B7) or mucous ceils alone (A1FS, A8E4) (10). dF508 mutation analysis. Genomic DNA was prepared from ZCFNPE14o- cells at different passages and PCR amplified with the GeneAmp kit. Oligonucleotide primers CF8C (5'-ATAGGAAACACCAATGATAT-3') or CF7C (5'-ATAGGAAACACCAAAGATGA-3')(antisense)(bp 1649-1668 of wtCFTR cDNA) and CF1B (5'-CCTTCTCTGTGAACCTCTATCA-3') (sense) were used for allele-specific amplification of AF508 or wild type CFTR DNA, respectively. The AAA in primer CF7C
FIG. 1. Morphologicand immunocytochemical characterization of ECFNPE14o- ceils at Passage 4.9 (4 passages before transfection with the pSVori-plasmid, 9 passages post-transfection), a, Light microscopy of growth clusters (islands) of cells after transfer into MEM supplemented with 10% FBS. Immunofluorescentstaining for the presence of b the SV40 large T-antigen, c keratin filaments characteristic of epithelial cells, and d junctional complex associated ceI1CAM120/80.
619
PROPERTIES OF A AF508 CF CELL LINE
a
70 6O z
50-
x
40-
,,-I II U. UJ
30-
=t
~,
";.
...": ~."
\\
"o',, "...,.',, FORS
H
........o ........ IONO .... o ....
A23187
20 10 0 0
l
I
I
I
I
I
I
1
2
3
4
5
6
7
TIME
5-
(MIN)
(n=9)
b
4-
~0 (1) 0 t'm
U--I
3-
T
2-
e-
o
(J 1 -
OFIG. 2. Measurement of cAMP-dependent (a) 36C1- efflux after exposure to forskolin (FOR, 10 ~ or after treatment with (a) A23187 (10 I.tM) and ionomycin (0.5 p.M) at 3 min after the start of the experiment. Aliquots were taken at 1-min intervals as described in the text. b, A summary of whole cell patch clamp analysis of individual cells after forskolin treatment. Values were calculated at 1 min after stimulation with forskolin. Error bars represent standard error of the mean (SEM) and n = the number of cells tested.
indicates the phenylalanine codon in CFTR in wild type exon 10. The AF508 mutation represents a 3 bp deletion of a CTI" (in primer CF7C this deletion is indicated in bold as AAG) to yield primer CF8C. DNA was amplified for 30 cycles as follows: denaturation for 30 s at 95 ° C, annealing for 30 s at 56 ° C, and extension 1 rain 30 s at 72 ° C, and then one additional annealing cycle is for 30 s at 56 ° C, and final extension for 8 min at 72 ° C. The PCR products were separated on 1% agarose gels. CFTR cDNA was analyzed as part of the DNA preparation described above. The PCR amplification was also allele-specific for either wild-type of AF508 CFTR sequences. The primary difference between the analysis of genomic DNA and cDNA is the use of oligonucleotide primers spanning intron-exon boundaries from exon 9 to 11. Using primers that span intronexon boundaries eliminates amplification of genomic DNA. PCR amplification using primers CF8B: 5'-CCATTAAAGAAAATATCATTGG-3'or CF7B: 5'-CCATFAAAGAAAATATCATCqT-3' (sense) and CF22K: 5'TTGqTAGCCATCAGTITACAG-3' (antisense) (base pair 1634-1925 of CFTR cDNA) yields a 290/293 bp product, respectively. The Cq'T in primer CFTB indicate the sequences in the wtCFTR that are deleted to give rise to the AF508 lesion. Primer CF8B is based on this deletion at the 3' end of the primer. The genomic DNA specific primer CF6: 5'-CCACATATCACTATATGCATGC-3' (antisense) was used in conjunction with the sense primers CF7B and CF8B to verify that there was no wt sequence associated with
genomic DNA. PCR products were separated by electrophoresis on 1% agarose gels and stained with ethidium bromide. Complementation with wtCFTR. Cells were transfected as described by CaPO4 precipitation with the pREP5/CFTR vector (24). Colonies of hygromycin B (300 IxM)-resistant cells were pooled and grown. The cells were then characterized genotypically for the presence of wtCFTR cDNA as described above. Ion transport characteristics were determined by whole cell patch clamp. Cl efflux. C1- transport was measured by radioactive 36C1- efflux. Cell were grown to confluence in MEM with 10% FBS at 37 ° or 26 ° C. After rinsing the cultures twice with 2 ml efflux buffer, fresh efflux buffer (1 ml) containing 2 I.tCi/ml 36C1- was added to each dish. Cultures were incubated for 2 h at 37 ° C and then washed by dipping into 200 ml efflux buffer for a wash time of 8 to 10 s. After washing, 1 ml efflux buffer was added, removed at 1-min intervals, and replaced with fresh buffer. At 3 min, efflux buffer containing 10/aM forskolin (Sigma, St. Louis, MO), 0.5 laM ionomycin (Sigma), or 10 /aM A23187 was added. Samples containing agonist were removed and replaced at 1-min intervals. Cells were extracted with 0.1 N HC1 (1 ml) overnight at 4 ° C, and the 36C1- remaining in the cells was determined. Samples were counted in scintillation cocktail. The percent efflux/1-min time point was calculated as follows: % efflux/min = (cpm for sample/total cpm in the cells for that minute). Whole-cellpatch clamp. Ceils were studied 1 to 4 days after plating on FN/ V/BSA-eoated glass coverslips. The dishes were mounted on the stage of an inverted microscope (Zeiss, Germany). The bath perfusion rate was approximately 20 ml/min corresponding to a bath exchange rate of twice per second. The standard bath solution contained (mM): NaC1 145; K2HPO 4 1.6; KH2PO4 0.4; CaClz 1.3; MgCI~ 1; D-glucose 5. A flowing KC1 electrode served as a reference and appropriate corrections for liquid-junction voltages have been made. The whole cell (wc) patch clamp method used has been described in detail (26). The patch pipettes were filled with a solution containing (mM): K+-gluconate 115; KC1 30; NaH2PO 4 0.4; Na=HPO 4 1.6; EGTA 1; CaCI2 0.5; MgCI~ 1; D-glucose 5; ATP 1. The pH was adjusted to 7.2 and Ca +2 activity in this solution was around 10- r M. The data were recorded using a LM EPC7 patch clamp amplifier (List, Darmstadt, Germany) and continuously displayed by a pen recorder. The access conductance was controlled in each experiment and was around 40 to 130 nS. The membrane voltage of the cells was recorded continuously using the current clamp mode of the patch clamp amplifier, and the wc-current was measured at regular intervals by clamping the membrane voltage to --+30 mV in steps of 10 inV. The wc-conductance (G) was calculated from the measured wc-current (I) and the applied clamp voltage (Vo). All experiments were carried out at 37 ° or 26 ° C. Secretagogues (ATP, bradykinin, and histamine) were obtained from Sigma and prepared fresh for each experiment from 1 mM stock solutions. Stock solutions were kept at - 2 0 ° C. Treatment doses for ATP, bradykinin, and histamine were 10/aM unless otherwise indicated. The hypotonic bath solution was an extracellular solution which was 145 mosmol due to removal of NaC1. The pipette solution was as described above.
TABLE 1 SECRETORY CELL-SPECIFIC ANTIGENS" Antibody
A1D3 AIEll B6G6 B6E8 B8C3 A1F8 A8E4 A2E7 A3B7
Staining
20-30% 25--40% 40-60% 10-25% 10-25% negative negative negative negative
strongly positive strongly positive medium to strongly positive strongly positive medium positive
In Vivo Localization
G, M, S G, M G, M G, M G, S M M S S
"In vitro staining of culture ZCFNPE14o- cells with secretory cell-specific antibodies. Antibodies are localized to goblet, serous, and mucous cells (G, M, S), goblet and mucous cells (G, M), goblet and serous cells (G, S), mucous cells alone (M), or serous cells alone (S) in vivo.
620
KUNZELMANNET AL. RESULTS
proportion of cells stained with the antibodies had characteristics that are distinctive of goblet cells (Table 1). Further analysis of ion transport by z6Cl- efflux (Fig. 2 a) and by whole cell patch clamp (Fig. 2 b) also indicated a lack of cAMPdependent C1- transport. However, the ceils did show a Ca+2-dependent C1- efflux (ionomycin or A23187) and whole cell conductances. Whole cell patch clamp analysis showed that both bradykinin and histamine increase whole cell conductance and membrane voltage (AVm) in a dose-dependent fashion (Fig. 3). Although the conductance showed a consistent increase for both bradykinin (10-lo to 10 6 M) and histamine (10 -s to 10 _4 M) (Fig. 3 a,b), the increase in V~ seemed to reach a plateau (Fig. 3 c,d). Whole cell patch clamp analysis showed that the increased whole cell conductance correlated with membrane hyperpolarization after exposure to bradykinin (0.1 ~tM) (Fig. 4 a), ATP (100 ~tM) (Fig. 4 b), or histamine (10 ~tM) (Fig. 4 c). The hyperpolarization by these agents have indicated activation of K ÷ conductance and to a smaller degree C1- conductance (21). Whole cell analysis of responses induced by ionomycin (0.1 ~tM, n = 2) also indicated an activation of K + current (unpublished observations). These studies show that the ECFNPE14o- cells have ion transport characteristics detected in other pSVori-transformed AF508 CF airway epithelial ceils (22) and that they have cell surface receptors implicated in regulation of Ca+Z-dependent ion transport
After transfection with the pSVori-plasmid, the cells were grown in serum-free MLHC8e medium for three subcultures and then transferred to MEM containing 10% FBS serum. Cells that were not transformed underwent squamous differentiation and ultimately peeled off the dish. All the colonies of growing transformed cells were pooled and expanded. The transformed cells tended to grow in small islands (Fig. 1 a) that have characteristically been associated with the retention of tight junctions (4,5). Staining with fluorescent-labeled antibodies to SV40 large T-antigen (Fig. 1 b), cytokeratins (Fig. 1 c), and ceI1CAM120/ 80 (Fig. 1 d) were used to assess transformation, epithelial origin, and the presence of junctional complexes indicative of tight junctions, respectively. To gain further insight into the potential origin of these cells, cultures were screened with secretory cell-specific antibodies. These antibodies have been shown to identify antigens that are associated with specific secretory cells in vivo (10). The staining that a significant portion of the cells were positive for antigens found in goblet and serous cells, goblet and mucous cells, and goblet, mucous, and serous cells (Table 1). Staining with antibodies specific for mucous or serous cells alone was negative and therefore suggests that the
a lOO
40A
b (n=4)
35-
~
30-
(n=5)
80
v
25-
~
60-
~
40-
20-
= "O o
15-
7 @
10
o
20-
5 0 10 -10
10 -9
10 -8
10 -7
10 -8
10
10 -7
E
5045 403530 25
10 -6
10 -5
10 -4
Histamine (tool/I)
Bradykinin (tool/I) C
d
50 45 4O
(n=5)
~
30
~ 2s E 20 > 15
20 15 10
5 0 -5
5 0
1
i
10 -10
i ILIHq
I
10 -9
q IIHN
I
I
10 -8
I llIHq
I
10 -7
Bradykinin (mol/I)
~ I[NIq
10 -6
'
10 -8
'['""1
'
10-7
' ......
I
'
''""q
10 -6
'
10 -5
'"'"1
10 -4
Histamine (mol/I)
FIG. 3. A dose-response curve for bradykinin (a and c) and histamine (b and d) induced increases in whole cell conductance and V m. Increased whole cell conductance (a and b) and membrane voltage (c and d) were determined after exposure to increasing doses of bradykinin (10-lo to 10-6 M) and histamine (10-s to 10 _4 M). Error bars indicate standard error of the mean (SEM) and n = the number of cells patched.
PROPERTIES OF A AF508 CF CELL LINE 40-
24-
(n=10) 30-
/
m 20-
0
10-
con
0-
-20 -
>
20-
Bradykinin
100 -
=
(n=4)
(n=15)
60-
~ 1240-
8
8
O
20-
4-
m
con
con
ATP
i
con
con
II II
II
-10 -20 ~"
-30
>
-40
E
-50
~
E >
bradykinin
con
b
-40-
-60 -
(n=4) -80
-70 -
con
con
II
-20 -
_L
-60 -
-6O
Histamine
0-
0
(n=13)
I'
80-
16-
-40-
a
621
con
ATP
C
con
con
Histamine
FIG. 4. Analysis of Ca+2-dependent El- transport as measured by whole cell patch clamp. Whole cell patch clamp analysis after exposure to Ca +2 agonists, a bradykinin (0.1 p.M), b ATP (100 W14),and c histamine (10 ~tM). Although the cells were unresponsive to cAMP agonists, they showed significant whole cell conductance (upper panel a,b, and c) and membrane hyperpolarization (lower panel a,b, and c). Error bars represent standard error of the mean (SEM) and n = the number of cells tested.
pathways (21). In addition to the intact Ca+2-dependent ion transport pathways, the cells are also able to regulate cell volume as is indicated by the increase in conductance after exposure to hypotonic (75 mM NaC1) solution (Fig. 5). Genotypic characterization of the cells by allele-specific PCR of genomic DNA indicated that the cells were homozygous for the AF508 CFTR mutation (Fig. 6). RT-PCR analysis of mRNA-derived cDNA showed that mutant CFTR mRNA was expressed in these cells, however, Northern hybridization analysis was unable to detect levels of CFTR mRNA sufficient to give a definitive signal (data not shown). The lack of detectable CFTR mRNA by Northern hybridization has been observed for most pSVori-transformed airway epithelial cell lines (4,6,13,22) with the notable exception of the highly differentiated normal cell line, 16HBE14o- (5). Previous studies indicated that cells expressing AF508 CFTR were able to show partial restoration of cAMP-dependent ion transport after growth at 26" C (7). Because this cell line is homozygous for the AF508 mutation it was possible to investigate the effect of temperature on CFTR function in an airway epithelial cell. The cells were tested for enhanced C1- secretion after growth at 26 ° C for 48 h with subsequent exposure to forskolin (10 ~tM) (Fig. 7 a). This was also confirmed by patch clamp analysis of C1- conductance (Fig. 7 b). There was no difference in responsiveness between growth at 26 ° or 37 ° C, nor was there any effect of carrying out the efflux analysis at 26 ° or 37 ° C (Fig. 7 a). The cells retained their responsiveness to ionomycin at all temperatures. To determine if there was anything unusual about these cells that would inhibit CFTR function or trafficking, the cells were transfected with a plasmid containing wtCFTR cDNA. This plasmid, pREP5CFTR, is Epstein-Barr virus (EBV)-based and seems to be primarily maintained as an episome (24). Cells were analyzed for the presence
of wtCFTR cDNA by amplification with primers that would require amplification across intron/exon boundaries (CF7B/CF22K) (Fig. 8 a). Analysis of genomic DNA-specific primers (CF8B/CF6) and (CF7B/CF6) also confirmed that the cells were AF508 homozygotes. RT-PCR analysis of RNA indicated the presence of wtCFTR mRNA in cells transfected with pREP5CFTR (data not shown). When the cells were tested for cAMP-dependent C1- transport by stimulation with forskolin, they showed a significant increase (twofold) in C1-
100
-
(n=10)
80-
cO tO C
60-
~6 40t-
O
0
20-
_
con
Cell Swelling
con
FIG. 5. Wholecell patch clamp analysis of swell-induced conductance after hypotonic shock. Error bars represent standard error of the mean (SEM) and n = the number of cells sampled.
622
KUNZELMANNET AL.
AF
N
I "
ZzO
:~ Q. Q.Q.
