BIOCHEMICAL
MEDICINE
AND
METABOLIC
BIOLOGY
47, 260-264 (1992)
BRIEF COMMUNICATION Sulfate Transport in Normal and Cystic Fibrosis Fibroblasts The glycoconjugate component of cystic fibrosis (CF) epithehal secretions is abnormally sulfated. Previous studies have suggested that some but not all CF fibroblasts express this secondary defect. We tested the hypothesis that the major CF mutation (AF,,/AF,,) is correlated with elevated sulfate transport, by measuring the rates of saturable and nonsaturable [“S&SO:- uptake in skin fibroblasts isolated from CF patients of known genotype. No significant differences were apparent between normal and CF fibroblasts. 8 19~ Academic Press. Inc.
Cystic fibrosis (CF) is the most common fatal genetic disease of the Caucasian population (1). While the disease is known to result from abnormal electrolyte transport, the pathophysiological link between this basic defect and the accumulation of dehydrated, abnormally synthesized, secretions is not well established. Recent developments in the molecular biology of CF have suggested that it can result from more than 100 different mutations (2), yet dehydrated secretions are a feature of all. Previous observations of abnormal sulfation of mucus glycoconjugates isolated from CF patients (3) have been verified by studies of isolated epithelia and epithelial cells in culture (4-6). The observations remain of great interest since oversulfation of mucins may influence both physicochemical properties of the mucus they constitute and persistence of such organisms as Staphylococcus aweuS and Pseudomonas aeruginosa in the airways. Further, an understanding of the mechanistic basis to oversulfation may indicate intracellular functions of the cystic fibrosis transmembrane conductance regulator (CFTR). The expression of the CF defect, or indeed the CFTR protein, in fibroblasts remains controversial (7-9.). However, while several studies have suggested that CAMP-dependent chloride fluxes may not be inducible in fibroblasts (lO,ll), others have suggested that sulfate uptake may be aberrant in some CF fibroblasts (12,13). It seemed valid to ask if genotypic variation between the fibroblast lines previously employed could have accounted for the phenotypic variation observed. The properties of the fibroblast plasma membrane sulfate transport system have been thoroughly defined (14), including its inhibition by the stilbene 4-acetamido-4isothiocyanatostilbene-2,2’-disulfonic acid (SITS) (13). These features have been incorporated in the standardized sulfate uptake assay employed. This work has been presented in preliminary form as a poster (15). 260 08854505192 $5.00 Copyright All rights
0 1992 by Academic Press. Inc. of reproduction in any form reserved
BRIEF COMMUNICATION
MATERIALS
261
AND METHODS
The source and routine maintenance of the fibroblast cultures has been described elsewhere (16). Fibroblasts were seeded to confluence in petri dishes (10 cm2, Costar, Cambridge, MA) based on P-hexosaminidase activity (17). At the start of the experiments, the cell layers were washed twice with 1 ml of PBS (Commonwealth Serum Laboratories, Melbourne, Vie.) and then preequilibrated for 50 min at 37°C in a 5% COJair environment with 150 mM NaCI, 4 mM Mgegluconate, 10 mM Hepes, 300 PM Na,SO,. After 50 min the dishes were transferred quickly to a 37°C water bath (open to the atmosphere) and the preincubation solution was discarded. The cell layers were washed rapidly with 1 ml of preequilibration solution. Exactly I ml of an uptake medium consisting of 150 mM NaCl, 4 mM Mgagluconate, 25 mM MES, 4.6 mM Tris, pH 5.5, Na2S04 (50, 70, or 114 PM at 5 &i/ml) was then added. Cells were exposed to the uptake medium for 1 min and then washed rapidly six times with 1.5 ml of ice-cold PBS. The uptake solution and the ice-cold PBS had been saturated with N2 for 15 min prior to the experiment to ensure a constant CO* gradient during transport experiments. Finally, the culture dishes were drained briefly and the cells solubilized with 0.1 M NaOH. To investigate the inhibition by SITS, it was added at 1 mM to cultures 15 min before the start of the experiment and again in the uptake buffer. Protein was measured by the BCA method (Pierce (18)). Scintillation counting was performed with LKB Optiphase HiSafe. Transport experiments were performed with a total of 13 fibroblast cell lines obtained from either the Department of Chemical Pathology at the Adelaide Children’s Hospital or the NIGMS Human Genetic Mutant Cell Repository. The seven CF lines were confirmed as AF508/AFS08 genotype by routine PCR analysis (19), kindly performed by P. Nelson. The cell lines came from fetuses (n = 2) and patients between the ages of 2 weeks and 18 years. Normal cell lines were chosen to encompass this range. All cultures were less than 20 passages. RESULTS AND DISCUSSION Transport studies suggest that little, if any, CFIR is expressed in fibroblasts, as opposed to epithelial cells (e.g., T,) (10,ll). However, PCR measurements suggest low level expression of CFTR with unknown biological significance (15). Given previous controversy over CF expression and sulfate uptake in fibroblasts, a question remained of whether genetic heterogeneity of the disease was at least partially responsible. Consistent with the work of others (13,14), [“‘S]SO:- uptake into fibroblasts was found to be rapid and primarily saturable. The major component of uptake could be described by saturation kinetics with a K, of 210 + 30 VM and V,,,,, of 370 r 90 pm01 min -’ mg protein-‘. A minor nonsaturable component was approximated by the uptake in the presence of 1 mM SITS. Both components were tested in the normal (n = 6) and CF (n = 7) cultures so as to exclude metabolic defects. The transport rates, standardized per unit culture protein, were not sig-
262
BRIEF COMMUNICATION
250
m
. .
200
B
150.
.E
E
1 “u s 3
0
0 l
loo.
a 0
l
*
0 0 50.
A
a
0
0
5b CONCENTRATION
IA0
OF SULPHATE
do
MM
FIG. 1. Uptake of Na2[?@O~ into fibroblasts as a function of extracellular Na$O, concentration. Fibroblasts (open squares, normal; closed squares, CF) were preequilibrated to 300 FM NaSO, for 50 min prior to addition of a radiolabeled uptake medium; between 5 and 10 &i/ml Na2[35S]S04, 50, 70, or 114 PM total Na$SO,. Uptake was for 1 min at 37°C stopped with ice-cold PBS and the cells were solubilized with 0.1 M NaOH. In other experiments, SITS (1 mM) was added during the last 15 min of the preincubation, and again during the uptake (open circles, normal; closed circles, CF).
nificantly different between the normals and the CF cultures at three Na2S04 concentrations, with or without SITS (Fig. 1). Increasing concentrations of Na2S04 appeared to increase the effectiveness of SITS as an inhibitor in both normal and CF cultures. We conclude that previous observations of elevated sulfate uptake (12,13) did not reflect the case for fibroblasts with the most common CF mutation, A&d AFm . It would be interesting to investigate cell lines with mutations in the
BRIEF COMMUNICATION
263
R domain of CFI’R, as these may express unusual anion selectivity or channel gating (20). ACKNOWLEDGMENTS This work was supported by the National and Medical Research Council as an Australian postdoctoral fellowship (G.S.H.), and the Channel 7 Children’s Medical Research Foundation (B.M.K.).
REFERENCES 1. Boat TF, Welsh MJ. Beaudet AL. Cystic Fibrosis. In The Metabolic Basis of Inherited Disease. (Striver CR, Beaudet AL, Sly WS, Valle D, Eds.). New York: McGraw-Hill, 1989, pp. 26492680. 2. Tsui L-C. Molecular genetics of cystic fibrosis. Annu Rev Hum Gene?., in press. 3. Boat TF, Cheng P-W, Iyer RN, Carlson DM. Polony I. Human respiratory tract secretions: Mucous glycoproteins of nonpurulent tracheobronchial secretions, and sputum of patients with bronchitis and cystic fibrosis. Arch Biochem Biophys 177:95-104. 1976. 4. Frates RC Jr, Kaizu ‘IT. Last JA. Mucus glycoproteins secreted by respiratory epithelial tissue from cystic fibrosis patients. Pediatr Res 17:30-34, 1983. 5. Boat TF. Kleinermen JI, Carlson DM, Maloney WH, Matthews LH. Human respiratory tract secretions: Mucous glycoproteins secreted by cultured nasal polyp epithelium from subjects with allergic rhinitis and cystic fibrosis. Annu Rev Respir Dis 110~428-441, 1974. 6. Cheng PW. Boat TF. Cranfill K, Yankaskas JR, Boucher RC. Increased sulfation of glycoconjugates by cultured nasal epithelial cells from patients with cystic fibrosis. J C/in Znvesf 84~68-72, 1989. 7. Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N. Chou JL. Drumm, ML, Ianuzzi MC, Collins FC, Tsui L-C. Identification of the cystic fibrosis gene: Cloning and characterization of complementary DNA. Science 245:1066-1073, 1989. 8. Lin PY, Gruenstein E. Identification of a defective CAMP-stimulated Cl-channel in cystic fibrosis fibroblasts. J Biol Chem 262:15345-15347, 1987. 9. Yoshimura K, Nakamura H, Trapnell BC, Chu C-S, Dalemans W, Pavirani A, Lecocq J-P, Crystal RG. Expression of the cystic fibrosis transmembrane conductance regulator gene in cells of nonepithelial origin. Nucleic Acid Res 19:5417-5423, 1991. lo., Squassoni E, Cabrini G, Berton G. CAMP dependent chloride conductance is not different in cystic fibrosis fibroblasts. Life Sci 46: 1265-1270, 1990. 11. Mastrocola T, Rugolo M. The response of chloride transport to cyclic AMP, calcium and hypotonic shock in normal and cystic fibrosis fibroblasts. Life Sci 46:1661-1669, 1990. 12. Wiesmann U, Neufeld EF. Metabolism of sulfated mucopolysaccharide in cultured fibroblasts from cystic fibrosis patients. J Pediatr 77:685-689, 1970. 13. Elgavish A, Meezan E. Increased sulfate uptake in skin fibroblasts isolated from cystic fibrosis patients. Biochem Biophys Res Commun 152~99%106, 1988. 14. Elgavish A, Meezan E. Sulfate transport in human lung fibroblast (IMR-90): effect of pH and anions. Am J Physiol256:C486-C494, 1989. 15. Rozaklis, T, King BM, Morris CP, Hopwood JJ, Harper GS. CFTR expression in skin fibroblasts: Individual or culture variation. Pediatr Pulmonol Suppl 6:169, 1991. 16. Taylor JA, Gibson GJ, Brooks DA, Hopwood JJ. Human N-acetylgalactosamine-4-sulphatase biosynthesis and maturation in normal, Maroteaux-Lamy and multiple-sulfatase-deficient fibroblasts. Biochem J 268~379-386, 1990. 17. Landegren U. Measurement of cell number by means of the endogenous enzyme hexosaminidase. Applications to detection of lymphokines and cell surface antigens. J Zmmuno/67:379-388, 1984. 18. Smith PK, Krohn RI, Hermanson GT, Mallia AK. Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. Measurement of protein using bicinchoninic acid. Anal Biochem 150~76-85,
1985.
264
BRIEF COMMUNICATION
19. Nelson PV, Carey WF, Morris CP. Gene amplification directly from Guthrie blood spots. Lancer 3361451-1452, 1990. 20. Rich DP, Gregory RJ, Anderson MP, Manavalan P., Smith AE, Welsh MJ. Effect of deleting the R domain on CFTR-generated chloride channels. Science 253~205-207, 1991. BARBARA M. KING TINA ROZAKLIS JOHN J. HOPWOOD GREGORY S. HARPER’ Department of Chemical Pathology Adelaide Children’s Hospital 72 King William Road North Adelaide, South Australia 5006 Australia Received January 10, 1992
’ To whom reprint requests should be addressed.
MEDICINE
AND
METABOLIC
BIOLOGY
47, 260-264 (1992)
BRIEF COMMUNICATION Sulfate Transport in Normal and Cystic Fibrosis Fibroblasts The glycoconjugate component of cystic fibrosis (CF) epithehal secretions is abnormally sulfated. Previous studies have suggested that some but not all CF fibroblasts express this secondary defect. We tested the hypothesis that the major CF mutation (AF,,/AF,,) is correlated with elevated sulfate transport, by measuring the rates of saturable and nonsaturable [“S&SO:- uptake in skin fibroblasts isolated from CF patients of known genotype. No significant differences were apparent between normal and CF fibroblasts. 8 19~ Academic Press. Inc.
Cystic fibrosis (CF) is the most common fatal genetic disease of the Caucasian population (1). While the disease is known to result from abnormal electrolyte transport, the pathophysiological link between this basic defect and the accumulation of dehydrated, abnormally synthesized, secretions is not well established. Recent developments in the molecular biology of CF have suggested that it can result from more than 100 different mutations (2), yet dehydrated secretions are a feature of all. Previous observations of abnormal sulfation of mucus glycoconjugates isolated from CF patients (3) have been verified by studies of isolated epithelia and epithelial cells in culture (4-6). The observations remain of great interest since oversulfation of mucins may influence both physicochemical properties of the mucus they constitute and persistence of such organisms as Staphylococcus aweuS and Pseudomonas aeruginosa in the airways. Further, an understanding of the mechanistic basis to oversulfation may indicate intracellular functions of the cystic fibrosis transmembrane conductance regulator (CFTR). The expression of the CF defect, or indeed the CFTR protein, in fibroblasts remains controversial (7-9.). However, while several studies have suggested that CAMP-dependent chloride fluxes may not be inducible in fibroblasts (lO,ll), others have suggested that sulfate uptake may be aberrant in some CF fibroblasts (12,13). It seemed valid to ask if genotypic variation between the fibroblast lines previously employed could have accounted for the phenotypic variation observed. The properties of the fibroblast plasma membrane sulfate transport system have been thoroughly defined (14), including its inhibition by the stilbene 4-acetamido-4isothiocyanatostilbene-2,2’-disulfonic acid (SITS) (13). These features have been incorporated in the standardized sulfate uptake assay employed. This work has been presented in preliminary form as a poster (15). 260 08854505192 $5.00 Copyright All rights
0 1992 by Academic Press. Inc. of reproduction in any form reserved
BRIEF COMMUNICATION
MATERIALS
261
AND METHODS
The source and routine maintenance of the fibroblast cultures has been described elsewhere (16). Fibroblasts were seeded to confluence in petri dishes (10 cm2, Costar, Cambridge, MA) based on P-hexosaminidase activity (17). At the start of the experiments, the cell layers were washed twice with 1 ml of PBS (Commonwealth Serum Laboratories, Melbourne, Vie.) and then preequilibrated for 50 min at 37°C in a 5% COJair environment with 150 mM NaCI, 4 mM Mgegluconate, 10 mM Hepes, 300 PM Na,SO,. After 50 min the dishes were transferred quickly to a 37°C water bath (open to the atmosphere) and the preincubation solution was discarded. The cell layers were washed rapidly with 1 ml of preequilibration solution. Exactly I ml of an uptake medium consisting of 150 mM NaCl, 4 mM Mgagluconate, 25 mM MES, 4.6 mM Tris, pH 5.5, Na2S04 (50, 70, or 114 PM at 5 &i/ml) was then added. Cells were exposed to the uptake medium for 1 min and then washed rapidly six times with 1.5 ml of ice-cold PBS. The uptake solution and the ice-cold PBS had been saturated with N2 for 15 min prior to the experiment to ensure a constant CO* gradient during transport experiments. Finally, the culture dishes were drained briefly and the cells solubilized with 0.1 M NaOH. To investigate the inhibition by SITS, it was added at 1 mM to cultures 15 min before the start of the experiment and again in the uptake buffer. Protein was measured by the BCA method (Pierce (18)). Scintillation counting was performed with LKB Optiphase HiSafe. Transport experiments were performed with a total of 13 fibroblast cell lines obtained from either the Department of Chemical Pathology at the Adelaide Children’s Hospital or the NIGMS Human Genetic Mutant Cell Repository. The seven CF lines were confirmed as AF508/AFS08 genotype by routine PCR analysis (19), kindly performed by P. Nelson. The cell lines came from fetuses (n = 2) and patients between the ages of 2 weeks and 18 years. Normal cell lines were chosen to encompass this range. All cultures were less than 20 passages. RESULTS AND DISCUSSION Transport studies suggest that little, if any, CFIR is expressed in fibroblasts, as opposed to epithelial cells (e.g., T,) (10,ll). However, PCR measurements suggest low level expression of CFTR with unknown biological significance (15). Given previous controversy over CF expression and sulfate uptake in fibroblasts, a question remained of whether genetic heterogeneity of the disease was at least partially responsible. Consistent with the work of others (13,14), [“‘S]SO:- uptake into fibroblasts was found to be rapid and primarily saturable. The major component of uptake could be described by saturation kinetics with a K, of 210 + 30 VM and V,,,,, of 370 r 90 pm01 min -’ mg protein-‘. A minor nonsaturable component was approximated by the uptake in the presence of 1 mM SITS. Both components were tested in the normal (n = 6) and CF (n = 7) cultures so as to exclude metabolic defects. The transport rates, standardized per unit culture protein, were not sig-
262
BRIEF COMMUNICATION
250
m
. .
200
B
150.
.E
E
1 “u s 3
0
0 l
loo.
a 0
l
*
0 0 50.
A
a
0
0
5b CONCENTRATION
IA0
OF SULPHATE
do
MM
FIG. 1. Uptake of Na2[?@O~ into fibroblasts as a function of extracellular Na$O, concentration. Fibroblasts (open squares, normal; closed squares, CF) were preequilibrated to 300 FM NaSO, for 50 min prior to addition of a radiolabeled uptake medium; between 5 and 10 &i/ml Na2[35S]S04, 50, 70, or 114 PM total Na$SO,. Uptake was for 1 min at 37°C stopped with ice-cold PBS and the cells were solubilized with 0.1 M NaOH. In other experiments, SITS (1 mM) was added during the last 15 min of the preincubation, and again during the uptake (open circles, normal; closed circles, CF).
nificantly different between the normals and the CF cultures at three Na2S04 concentrations, with or without SITS (Fig. 1). Increasing concentrations of Na2S04 appeared to increase the effectiveness of SITS as an inhibitor in both normal and CF cultures. We conclude that previous observations of elevated sulfate uptake (12,13) did not reflect the case for fibroblasts with the most common CF mutation, A&d AFm . It would be interesting to investigate cell lines with mutations in the
BRIEF COMMUNICATION
263
R domain of CFI’R, as these may express unusual anion selectivity or channel gating (20). ACKNOWLEDGMENTS This work was supported by the National and Medical Research Council as an Australian postdoctoral fellowship (G.S.H.), and the Channel 7 Children’s Medical Research Foundation (B.M.K.).
REFERENCES 1. Boat TF, Welsh MJ. Beaudet AL. Cystic Fibrosis. In The Metabolic Basis of Inherited Disease. (Striver CR, Beaudet AL, Sly WS, Valle D, Eds.). New York: McGraw-Hill, 1989, pp. 26492680. 2. Tsui L-C. Molecular genetics of cystic fibrosis. Annu Rev Hum Gene?., in press. 3. Boat TF, Cheng P-W, Iyer RN, Carlson DM. Polony I. Human respiratory tract secretions: Mucous glycoproteins of nonpurulent tracheobronchial secretions, and sputum of patients with bronchitis and cystic fibrosis. Arch Biochem Biophys 177:95-104. 1976. 4. Frates RC Jr, Kaizu ‘IT. Last JA. Mucus glycoproteins secreted by respiratory epithelial tissue from cystic fibrosis patients. Pediatr Res 17:30-34, 1983. 5. Boat TF. Kleinermen JI, Carlson DM, Maloney WH, Matthews LH. Human respiratory tract secretions: Mucous glycoproteins secreted by cultured nasal polyp epithelium from subjects with allergic rhinitis and cystic fibrosis. Annu Rev Respir Dis 110~428-441, 1974. 6. Cheng PW. Boat TF. Cranfill K, Yankaskas JR, Boucher RC. Increased sulfation of glycoconjugates by cultured nasal epithelial cells from patients with cystic fibrosis. J C/in Znvesf 84~68-72, 1989. 7. Riordan JR, Rommens JM, Kerem B, Alon N, Rozmahel R, Grzelczak Z, Zielenski J, Lok S, Plavsic N. Chou JL. Drumm, ML, Ianuzzi MC, Collins FC, Tsui L-C. Identification of the cystic fibrosis gene: Cloning and characterization of complementary DNA. Science 245:1066-1073, 1989. 8. Lin PY, Gruenstein E. Identification of a defective CAMP-stimulated Cl-channel in cystic fibrosis fibroblasts. J Biol Chem 262:15345-15347, 1987. 9. Yoshimura K, Nakamura H, Trapnell BC, Chu C-S, Dalemans W, Pavirani A, Lecocq J-P, Crystal RG. Expression of the cystic fibrosis transmembrane conductance regulator gene in cells of nonepithelial origin. Nucleic Acid Res 19:5417-5423, 1991. lo., Squassoni E, Cabrini G, Berton G. CAMP dependent chloride conductance is not different in cystic fibrosis fibroblasts. Life Sci 46: 1265-1270, 1990. 11. Mastrocola T, Rugolo M. The response of chloride transport to cyclic AMP, calcium and hypotonic shock in normal and cystic fibrosis fibroblasts. Life Sci 46:1661-1669, 1990. 12. Wiesmann U, Neufeld EF. Metabolism of sulfated mucopolysaccharide in cultured fibroblasts from cystic fibrosis patients. J Pediatr 77:685-689, 1970. 13. Elgavish A, Meezan E. Increased sulfate uptake in skin fibroblasts isolated from cystic fibrosis patients. Biochem Biophys Res Commun 152~99%106, 1988. 14. Elgavish A, Meezan E. Sulfate transport in human lung fibroblast (IMR-90): effect of pH and anions. Am J Physiol256:C486-C494, 1989. 15. Rozaklis, T, King BM, Morris CP, Hopwood JJ, Harper GS. CFTR expression in skin fibroblasts: Individual or culture variation. Pediatr Pulmonol Suppl 6:169, 1991. 16. Taylor JA, Gibson GJ, Brooks DA, Hopwood JJ. Human N-acetylgalactosamine-4-sulphatase biosynthesis and maturation in normal, Maroteaux-Lamy and multiple-sulfatase-deficient fibroblasts. Biochem J 268~379-386, 1990. 17. Landegren U. Measurement of cell number by means of the endogenous enzyme hexosaminidase. Applications to detection of lymphokines and cell surface antigens. J Zmmuno/67:379-388, 1984. 18. Smith PK, Krohn RI, Hermanson GT, Mallia AK. Gartner FH, Provenzano MD, Fujimoto EK, Goeke NM, Olson BJ, Klenk DC. Measurement of protein using bicinchoninic acid. Anal Biochem 150~76-85,
1985.
264
BRIEF COMMUNICATION
19. Nelson PV, Carey WF, Morris CP. Gene amplification directly from Guthrie blood spots. Lancer 3361451-1452, 1990. 20. Rich DP, Gregory RJ, Anderson MP, Manavalan P., Smith AE, Welsh MJ. Effect of deleting the R domain on CFTR-generated chloride channels. Science 253~205-207, 1991. BARBARA M. KING TINA ROZAKLIS JOHN J. HOPWOOD GREGORY S. HARPER’ Department of Chemical Pathology Adelaide Children’s Hospital 72 King William Road North Adelaide, South Australia 5006 Australia Received January 10, 1992
’ To whom reprint requests should be addressed.
