EXPERIMENTAL
CELL RESEARCH
195,
194-198
(1991)
Secretion of Lysosomal Hydrolases by Cultured Human Amnion Epithelial Cells BRUNA Department
SCAGGIANTE,MARINACOMELLI,ANDDOMENICOROMEO'
of Biochemistry,
Biophysics
and Macromolecular
Clniurrsity
of Tries&,
34127
Trieste,
Italy
lial cells from the other cell types of amnion, mainly fibroblasts, and to study whether, once maintained in culture, they are capable of releasing lysosomal enzymes into the culture medium. This investigation has been performed by establishing primary microcultures of amniotic epithelial cells in a serum-free medium, which has permitted a kinetic and quantitative analysis of the secretion of some hydrolases.
Primary microcultures of human amnion epithelial cells were established, starting from sterile term placentae. Over a period of 1 week in culture, the epithelial cells release into the extracellular medium substantial amounts of some lysosomal hydrolases, such as sphingomyelinase, N-acetyl-&glucosaminidase, cu-fucosidase, P-glucuronidase, cy-mannosidase, and arylsulfatase. Judging from experiments conducted with the protein synthesis inhibitor, cycloheximide, the enzymes released are not newly synthesized forms, but very likely derive from lysosomes. The constitutive secretion of lysosomal enzymes, coupled with lack of immunogenicity, makes amnion epithelial cells a convenient source of enzymes for implantation in attempts of enzyme replacement therapies. c issi Academic PRSS. 1~.
MATERIALS
AND
METHODS
Establishment of amniotic q%thelial cell culturrs. Human term placentae, derived from caesarian sections performed on healthy women, were supplied by the Obstetric Unit of the Children’s Hospital of Trieste. IJnder sterile conditions, the placenta was rinsed with isotonic saline solution and the amnion was gently peeled off from the underlying chorion. The amniotic tissue was placed in a petri dish containing an isotonic saline solution to remove residual blood clots adherent to the inner surface. The membrane was then finely minced with scissors to obtain a homogeneous tissue suspension. After centrifugation at SOOg for 10 min, the tissue fragments were suspended in 100 ml of PBS without calcium and magnesium, containing 0.1% trypsin (w/v), 0.03% collagenase type II (w/v), 100 IIJ/ml penicillin, and 100 fig/ml streptomycin (Seromed, Biochrom KG, Berlin, Germany) and incubated at 37°C in a shaking water-bath with gentle agitation. At 30.min intervals, the suspension of detached cells was collected, centrifuged at 2OOg for 10 min, and resuspended in culture medium Ham’s F-12 containing 10% FCS, 4 mM L-glutamine, 100 IU/ml penicillin, and 100 pglml streptomycin. The digestion of amniotic membrane fragments was continued until complete tissue disaggregation. Each amnion yielded l-2 X 10” cells, which were seeded at 0.5 X 106/ml in 150 cm2 Aasks and maintained in culture for at least 24 h to monitor fibroblast contamination. Fibroblasts always displayed the most rapid and strongest adhesion to the substratum. A subpopulation of epithelial cells also firmly adhered to the plastic surface. However, there was another vital subpopulation which showed a weak tendency to adhere and whose proportion appeared to increase with the age of the placenta. The two subpopulations of epithelial cells were identified by their reaction with a mouse antibody against cytokeratin 19 followed by a stain with anti-mouse immunoglobulin sheep IgG, conjugated with Texas Red (Amersham, Buckinghamshire, UK). Short and repeated treatments with 0.05% trypsin and 0.02% EDTA caused a selective and sequential detachment of the weakly adherent cells, the firmly adherent ones, and the fibroblasts, respectively. The subpopulation of firmly adherent epithelial cells was collected, centrifuged at 2OOg for 10 min, and counted in a Thoma chamber. Cells (2 x IO”) were then seeded in 2-cm* microtiter plates and conditioned with I ml of Ham’s F12 medium containing 50 rig/ml EGF (Collaborative Research, Inc., Lexington, MA), 0.5 @g/ml hydrocortisane, 0.24 IU/ml insulin, 25 @g/ml transferrin (Sigma, St. Louis, MO),
INTRODUCTION Among fetal tissues, human amnion has gained in recent years more interest as a dynamic tissue that takes part in metabolic regulations than as an immunological barrier or a mechanical support to the fetus. Some of these regulations are carried out by a selective release of a variety of proteins, which may exert critical functions during and/or at the end of pregnancy [l-lo]. For example, the secretion of lysosomal enzymes appears to play an important role in labor [B]. Secretion of specific proteins by amniotic tissue in culture may also be exploited for practical applications. At least in part, this has been the rationale for allograft implantations of amniotic membrane as a source of lysosomal enzymes in attempts of replacement therapies [ll]. However, the amniotic tissue is rather complex and contains several cell types, of which only the epithelial ones are relevant for implantations due to the absence of histocompatibility antigens on their surface [X2]. Thus we thought it important to separate the epithei To whom correspondence and reprint requests should be addressed at Dipartimento di Biochimica, Biofisica e Chimica delle Macromolecole, Universith di Trieste, via A. Valerio, 22, 34127 Trieste, Italy. 0014.4827/91 $3.00 Copyright Q 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Chemistry,
194
LYSOSOMAL
ENZYME
SECRETION
4 mA4 I,-glutamine, 100 I‘CJ/ml penicillin, 100 @g/ml streptomycin, and 20 mA4 Hepes, pH 7.36. Cell viability was evaluated by trypan blue exclusion tests. DNA determination. To determine the cell number in microcultures, given amounts of epithelial cells in control wells were solubilized with 0.1% SDS and the DNA content was measured by combining the methodologies described by Clifford et al. [13] and by Labarca and Paigen [14] and using calf thymus DNA (Sigma) as standard. Enzyme assays. Enzymatic activities were measured in both the culture medium and cells. These were solubilized at 37°C for 30 min in 0.25 ml of a low ionic extraction buffer containing 0.2% Triton X-100, 60 mM KCl, 1 mM EDTA, 2 mM EGTA, 1 mM cysteine, 10 mM ATP, and 40 mM imidazole, pH 7.0. Most lysosomal enzyme activities were assayed employing 4. methyl-umbelliferyl derivatives as substrates (Sigma). In a final volume of 0.5 ml, the assay mixtures contained Triton X-100 from 0.04 to 0.07cC and had the following composition: for N-acetyl-@-glucosaminidase (&GAM), 0.64 mM substrate, 50 mM citrate buffer, pH 4.5; for d-glucuronidase (B-CUR), 0.32 mM substrate, 50 mM acetate buffer, pH i3.8, 0.05% bovine serum albumin; for fi-galactosidase (dGAL), 0.5 mM substrate, 50 mM acetate buffer, pH 4.5; for p-glucosidase (&GLUC), 0.36 mA4 substrate, 200 mM acetate buffer, pH 5.5, 1.2? Na-taurocolate; for tu-fucosidase (tu-FUC), 0.6 mM substrate, 50 mA4 acetate buffer, pH 5.0; for n-mannosidase (n-MAN), 3.2 mA4 substrate, 50 mM citrate buffer, pH 4.2; for n-galactosidase (a-GAL), 3.2 mA4 substrate, 50 mM acetate buffer, pH 5; for a-glucosidase (aGLUC), 0.5 mM substrate, 50 mM citrate buffer, pH 4.5; and for arylsulfatase (ARYL), 5 mM substrate, 50 mM acetate buffer, pH 5.5. After a 30- to 60.min incubation at 37”C, the reactions were stopped with 1.5 ml of 1 M glycine/NaOH buffer, pH 10.45, and the fluorescence of umhelliferone was measured at X,, = 360 nm and X,, - 450 nm. Acid phosphatase (AC PH) was assayed by a spectrophotometric method utilizing as substrate 2 mlM 4-nitrophenylphosphate (Sigma) in 0.1 ml of final volume with 50 mM acetate buffer, pH 4.5, 150 mM serine, 1 mM MgCI,, and 0.5% Triton X-100. After a 30-min incubation at 37”C, the reaction was stopped with 1 ml of 1 N NaOH and the reaction product was read at 405 nm. Sphingomyelinase activity (SPHINGO) was assayed by a radioisotopic method employing the natural substrate N-methyl-‘4C-sphingomyelin (Amersham, Buckinghamshire, UK). The substrate was prepared by repeated sonications (up to 30 min) of a mixture of radioactive and nonradioactive sphingomyelin in a ratio of 1:66, suspended in 2% Triton X-100 with 1 mM NaN,. The enzyme assay was carried out in a volume of 0.2 ml, with 0.1 M acetate buffer, pH 5.5, 0.05 mM magnesium acetate, 0.2% Triton X-100,67 nmol of substrate (140,000 cpm). After a 3-h incubation at 37”C, the reaction was stopped with 1 ml of cold 10% (w/v) bovine serum albumin and 0.2 ml of 50% (w/v) trichloracetic acid (TCA). The supernatant solutions, obtained by centrifugation at 1OOOOg for 10 min, were decanted and the precipitates were resuspended in 1 ml of cold 10% TCA and centrifuged. One milliliter of combined supernatants, containing W-labeled reaction products, was mixed with 10 ml of scintillator mixture (Aqualuma) for radioactivity determination. Blanks were between 50-100 cpm. For the ahove enzymes, one unit was defined as micromoles of reaction product generated per hour. The LDH activity was assayed in cuvette with 0.6 ml of 0.1 M sodium phosphate/HCl buffer, pH 7.5, containing 0.27 mM NADH, 0.02% Triton X-100, and sample up to 0.95 ml. The reaction was started with 50 ~1 of 32 mA4 sodium pyruvate. The decrease in absorbance was read at 340 nm with a lin-log recorder. One unit was defined as millimoles of reaction product generated per minute. Experiments with cycloheximide. Epithelial cells (0.5 X 104) were seeded in microtiter plates using as culture medium MEM with 10% FCS, 4 mA4 L-glutamine, 100 IU/ml penicillin, and 100 pg/ml streptomycin. After 24 h, the medium was changed and the cells were conditioned in serum-free MEM for 24 h. Cells were then exposed to
BY
EPITHELIAL
CELLS
195
serum-free MEM without leucine for 6-8 h prior to the addition of 1 FCi of L-[4,5-3H]leucine (Amersham) per well and 5 pg/ml cycloheximide. After 24-h incubation, the media were removed and cells were solubilized with 100 ~1 of 1% SDS. Proteins were then precipitated with 800 ~1 of 10% TCA and filtered on Millipore filters, which were rinsed with 10 ml of 5% TCA and 5 ml of absolute ethanol. The incorporation of [“Hlleucine into proteins was evaluated by scintillation counting. To test the effects of cycloheximide on p-GAM and 0-GIIR activities, 1 X lo6 cells were seeded in Ham’s F-12 medium containing 10% FCS, 4 mA4 L-glutamine, 100 IU/ml penicillin, and 100 pg/ml streptomycin. After 24 h the medium was changed and the cells were conditioned for an additional 24 h in Ham’s F-12 serum-free medium. The cells were then exposed to serum-free medium with or without 5 pg/ ml cycloheximide, incubated for 24 h, and tested for the two hydrolase activities.
RESULTS
Since lysosomal enzyme activities in cell culture may vary according to the growth phase [ 151 and the spreading of cells during attachment on culture dishes may facilitate the exocytosis of lysosomal content [16], the amnion epithelial cells were seeded at nearly confluence density and maintained in culture for 24 h before beginning the experiments. Furthermore, to avoid potential interferences of serum on enzymatic assays, microcultures were established in serum-free conditions. Specific enzyme activities within the cells and in the culture media were referred to cell number (per lo6 cells), because enzyme release may change the total cellular proteins. The number of epithelial cells in microculture was established by measuring the DNA content per well and comparing it with a calibration curve. In 21 experiments the DNA content per cell was estimated to be 15.1 & 0.59 pg (mean -t SEM). The quality of the assays performed was tested with parallel cultures of human fibroblasts, which provided an average value of 10 pg DNA/cell, consistent with data reported in the literature [ 171. To test the potential secretion of lysosomal hydrolases by amnion epithelial cells, we first followed the release of two lysosomal enzyme markers, /3-GAM and P-GUR, for a week at Days 1,4, and 7. At Days 1 and 4, cells which were going to be tesbed at Day 4 and 7, respectively, were exposed to fresh culture medium. As shown in Table 1, the epithelial cells may release about 12% of the total activities of P-GAM and /3-GUR after 24 h in culture, and from about 15 to 17% of total activities during two subsequent periods of 3 days. This process appears to be a true secretion, since concomitantly there is a very low or undetectable release of cytosolic LDH in the extracellular media. The secretion of P-GAM was also evaluated in the absence of EGF and hydrocortisone in the serum-free medium. Up to Day 4, the release of the lysosomal enzyme was virtually identical to the values found above, thereby excluding a stimulatory effect of EGF and/or
196
SCAGGIANTE,
TABLE Secretion glucuronidase
of N-acetyl-fi-glucosaminidase (P-GUR) from Human Enzyme
activity
IN
1 4 7
745 + 121* 680 AZ 118* 697 i 93*
AND
ROMEO
1
@-GUR
Amnion (mU/106
(@-GAM) and @Epithelial Cells
i-
cells) &GUR
$-GAM Days of culture
COMELLI,
95 Secretion 11.0
-t 0.8
14.2
+ 2.0
14.6 k 1.6
IN
5% Secretion
9.8 -+ 0.7* 10.4
+ 0.4*
11.8
t
1.s*
12.6 k 1.6 17.5 * 4.2 16.7 k 2.7
Note. At Days 1 and 4, cells which were going to be tested at Day 4 and 7, respectively, were exposed to fresh culture medium. Results are means + SEM for 6 to 13 experiments. R Secretion was calculated as I OIJT/(OUT + IN) j X 100. The percentage of total LDH activity found extracellularly at the same days of culture were 0.4 k 0.2, 0.5 + 0.5. and I.7 2 1.1 (means k SEM for 8 to 18 determinations). * P > 0.05 (Mann-Witney’s test) for differences among enzyme activities.
1
4
7 DAYS
1
4
$
OF CULTURE
FIG. 1. Extracellular and intracellular N-acetyl-&glucosaminidase (+GAM) and b-glucuronidase (8.GUR) activities in steady state conditions with epithelial cells maintained in culture up to 7 days. The activities are means k SEM for 12 to 14 determinations of fiGAM and for 8 to 12 determinations of 8-GUR.
hydrocortisone on secretion. Thereafter, however, cell survival rapidly diminished as revealed by an increasing recovery of LDH in the medium. Levels of extracellular lysosomal enzymes depend on the rate of secretion as well as on the rate of endocytothe seventh day of culture, the lysosomal enzymes studsis. To study the release of enzymes in a steady-state condition we then maintained the cells in culture for 1 ied may be divided into four classes.The first group with week, without changing the culture medium. Over this the highest rate of secretion (from 19 to 25% of the total includes P-GAM, cr-FUC, and period we did not observe any significant cell damage as enzyme activities) SPHINGO; enzymes of the second group, such as /Jrevealed by dye exclusion tests and by low levels of cytoGUR, (r-MAN, and ARYL, are present in the media at solic LDH in the culture medium (at the seventh day the levels of 10 to 14% of the total activities; the third group extracellular LDH ranged from 2 to 3.5% of the total includes AC PH, LU-GLUC, and Q-GAL, which are recell activity). The results illustrated in Fig. 1 show the release of the two glycosidases into the extracellular medium. For P-GAM, the amount of extracellular enzyme gradually increases to 25% of the total enzyme activity TABLE 2 over a period of 7 days, whereas for fi-GUR the maximal Effect of Cycloheximide on Protein Synthesis and on Nextracellular activity (15% of total) is reached on the acetyl-P-glucosaminidase (fi-GAM) and P-glucuronidase (pGUR) Activities in Cultured Amnion Epithelial Cells (IN) and fourth day. Their Bathing Media (OUT) To establish whether the released enzymes are newly synthesized or are products previously stored in lysoEnzyme activities somes, we then cultured cells in the presence or absence (mU/lO” cells) of 5 pg/ml cycloheximide for 24 h. At this concentration, P-GAM /SGUII [“H]Leucine cycloheximide inhibits by about 85% the incorporation incorporation of [3H]leucine into cellular protein and causes a deIN OUT OUT (cpm) IN crease of about 20% of the intracellular and of 16 to 30% of the extracellular activities of the two lysosomal hy- Control 80037 678 + 61 50.5 2 4.5 21.7 f 5.: 2.7 + 0.5 11440 540 i 50 42.4 -t 5.2 16.8 f 4.7 1.9 t 0.5 drolases (Table 2). However, these differences among Cycloheximide enzyme activities in control and treated samples do not Note. Cells were exposed to serum-free MEM without leucine prior appear to be statistically significant. to the addition of [aH]leucine and cycloheximide (5 fig/ml). After 24 To complement our studies on the appearance of /3- h, media were removed and radioactivity was measured in proteins. In parallel experiments, conducted in GAM and P-GUR in the cell bathing media, we also as- TCA-precipitable F-12 medium and without the addition of [3H]leucine, the sayed other lysosomal enzymes. Figure 2 illustrates the HAM’S activities of &GAM and B-GUR were evaluated in cells and bathing results of some experiments demonstrating a heterogemedia (see Materials and Methods). Results are means +- SEM for neous secretion of lysosomal hydrolases by amnion epi- four experiments. P f 0.05 (Mann-Witney’s test) for differences thelial cells. On the basis of the extracellular activity on among pairs of enzyme activities (controls vs cycloheximide).
LYSOSOMAL 20-
ENZYME
SECRETION
. a-FUC
IO-
t? 2
; nz
20-
o”
, ~-GAL
.a-GAL IAAC PH A a-GLUC
o P-GLUC
IO’
DAYS
OF
CULTURE
FIG. 2. Activities of sphingomyelinase (SPHINGO), cu-fucosidase (n-FUC), ru-mannosidase ( 3): SPHINGO (3), 12.0 i- 0.2; (u-FUC (2), 110.3;
CELL RESEARCH
195,
194-198
(1991)
Secretion of Lysosomal Hydrolases by Cultured Human Amnion Epithelial Cells BRUNA Department
SCAGGIANTE,MARINACOMELLI,ANDDOMENICOROMEO'
of Biochemistry,
Biophysics
and Macromolecular
Clniurrsity
of Tries&,
34127
Trieste,
Italy
lial cells from the other cell types of amnion, mainly fibroblasts, and to study whether, once maintained in culture, they are capable of releasing lysosomal enzymes into the culture medium. This investigation has been performed by establishing primary microcultures of amniotic epithelial cells in a serum-free medium, which has permitted a kinetic and quantitative analysis of the secretion of some hydrolases.
Primary microcultures of human amnion epithelial cells were established, starting from sterile term placentae. Over a period of 1 week in culture, the epithelial cells release into the extracellular medium substantial amounts of some lysosomal hydrolases, such as sphingomyelinase, N-acetyl-&glucosaminidase, cu-fucosidase, P-glucuronidase, cy-mannosidase, and arylsulfatase. Judging from experiments conducted with the protein synthesis inhibitor, cycloheximide, the enzymes released are not newly synthesized forms, but very likely derive from lysosomes. The constitutive secretion of lysosomal enzymes, coupled with lack of immunogenicity, makes amnion epithelial cells a convenient source of enzymes for implantation in attempts of enzyme replacement therapies. c issi Academic PRSS. 1~.
MATERIALS
AND
METHODS
Establishment of amniotic q%thelial cell culturrs. Human term placentae, derived from caesarian sections performed on healthy women, were supplied by the Obstetric Unit of the Children’s Hospital of Trieste. IJnder sterile conditions, the placenta was rinsed with isotonic saline solution and the amnion was gently peeled off from the underlying chorion. The amniotic tissue was placed in a petri dish containing an isotonic saline solution to remove residual blood clots adherent to the inner surface. The membrane was then finely minced with scissors to obtain a homogeneous tissue suspension. After centrifugation at SOOg for 10 min, the tissue fragments were suspended in 100 ml of PBS without calcium and magnesium, containing 0.1% trypsin (w/v), 0.03% collagenase type II (w/v), 100 IIJ/ml penicillin, and 100 fig/ml streptomycin (Seromed, Biochrom KG, Berlin, Germany) and incubated at 37°C in a shaking water-bath with gentle agitation. At 30.min intervals, the suspension of detached cells was collected, centrifuged at 2OOg for 10 min, and resuspended in culture medium Ham’s F-12 containing 10% FCS, 4 mM L-glutamine, 100 IU/ml penicillin, and 100 pglml streptomycin. The digestion of amniotic membrane fragments was continued until complete tissue disaggregation. Each amnion yielded l-2 X 10” cells, which were seeded at 0.5 X 106/ml in 150 cm2 Aasks and maintained in culture for at least 24 h to monitor fibroblast contamination. Fibroblasts always displayed the most rapid and strongest adhesion to the substratum. A subpopulation of epithelial cells also firmly adhered to the plastic surface. However, there was another vital subpopulation which showed a weak tendency to adhere and whose proportion appeared to increase with the age of the placenta. The two subpopulations of epithelial cells were identified by their reaction with a mouse antibody against cytokeratin 19 followed by a stain with anti-mouse immunoglobulin sheep IgG, conjugated with Texas Red (Amersham, Buckinghamshire, UK). Short and repeated treatments with 0.05% trypsin and 0.02% EDTA caused a selective and sequential detachment of the weakly adherent cells, the firmly adherent ones, and the fibroblasts, respectively. The subpopulation of firmly adherent epithelial cells was collected, centrifuged at 2OOg for 10 min, and counted in a Thoma chamber. Cells (2 x IO”) were then seeded in 2-cm* microtiter plates and conditioned with I ml of Ham’s F12 medium containing 50 rig/ml EGF (Collaborative Research, Inc., Lexington, MA), 0.5 @g/ml hydrocortisane, 0.24 IU/ml insulin, 25 @g/ml transferrin (Sigma, St. Louis, MO),
INTRODUCTION Among fetal tissues, human amnion has gained in recent years more interest as a dynamic tissue that takes part in metabolic regulations than as an immunological barrier or a mechanical support to the fetus. Some of these regulations are carried out by a selective release of a variety of proteins, which may exert critical functions during and/or at the end of pregnancy [l-lo]. For example, the secretion of lysosomal enzymes appears to play an important role in labor [B]. Secretion of specific proteins by amniotic tissue in culture may also be exploited for practical applications. At least in part, this has been the rationale for allograft implantations of amniotic membrane as a source of lysosomal enzymes in attempts of replacement therapies [ll]. However, the amniotic tissue is rather complex and contains several cell types, of which only the epithelial ones are relevant for implantations due to the absence of histocompatibility antigens on their surface [X2]. Thus we thought it important to separate the epithei To whom correspondence and reprint requests should be addressed at Dipartimento di Biochimica, Biofisica e Chimica delle Macromolecole, Universith di Trieste, via A. Valerio, 22, 34127 Trieste, Italy. 0014.4827/91 $3.00 Copyright Q 1991 by Academic Press, Inc. All rights of reproduction in any form reserved.
Chemistry,
194
LYSOSOMAL
ENZYME
SECRETION
4 mA4 I,-glutamine, 100 I‘CJ/ml penicillin, 100 @g/ml streptomycin, and 20 mA4 Hepes, pH 7.36. Cell viability was evaluated by trypan blue exclusion tests. DNA determination. To determine the cell number in microcultures, given amounts of epithelial cells in control wells were solubilized with 0.1% SDS and the DNA content was measured by combining the methodologies described by Clifford et al. [13] and by Labarca and Paigen [14] and using calf thymus DNA (Sigma) as standard. Enzyme assays. Enzymatic activities were measured in both the culture medium and cells. These were solubilized at 37°C for 30 min in 0.25 ml of a low ionic extraction buffer containing 0.2% Triton X-100, 60 mM KCl, 1 mM EDTA, 2 mM EGTA, 1 mM cysteine, 10 mM ATP, and 40 mM imidazole, pH 7.0. Most lysosomal enzyme activities were assayed employing 4. methyl-umbelliferyl derivatives as substrates (Sigma). In a final volume of 0.5 ml, the assay mixtures contained Triton X-100 from 0.04 to 0.07cC and had the following composition: for N-acetyl-@-glucosaminidase (&GAM), 0.64 mM substrate, 50 mM citrate buffer, pH 4.5; for d-glucuronidase (B-CUR), 0.32 mM substrate, 50 mM acetate buffer, pH i3.8, 0.05% bovine serum albumin; for fi-galactosidase (dGAL), 0.5 mM substrate, 50 mM acetate buffer, pH 4.5; for p-glucosidase (&GLUC), 0.36 mA4 substrate, 200 mM acetate buffer, pH 5.5, 1.2? Na-taurocolate; for tu-fucosidase (tu-FUC), 0.6 mM substrate, 50 mA4 acetate buffer, pH 5.0; for n-mannosidase (n-MAN), 3.2 mA4 substrate, 50 mM citrate buffer, pH 4.2; for n-galactosidase (a-GAL), 3.2 mA4 substrate, 50 mM acetate buffer, pH 5; for a-glucosidase (aGLUC), 0.5 mM substrate, 50 mM citrate buffer, pH 4.5; and for arylsulfatase (ARYL), 5 mM substrate, 50 mM acetate buffer, pH 5.5. After a 30- to 60.min incubation at 37”C, the reactions were stopped with 1.5 ml of 1 M glycine/NaOH buffer, pH 10.45, and the fluorescence of umhelliferone was measured at X,, = 360 nm and X,, - 450 nm. Acid phosphatase (AC PH) was assayed by a spectrophotometric method utilizing as substrate 2 mlM 4-nitrophenylphosphate (Sigma) in 0.1 ml of final volume with 50 mM acetate buffer, pH 4.5, 150 mM serine, 1 mM MgCI,, and 0.5% Triton X-100. After a 30-min incubation at 37”C, the reaction was stopped with 1 ml of 1 N NaOH and the reaction product was read at 405 nm. Sphingomyelinase activity (SPHINGO) was assayed by a radioisotopic method employing the natural substrate N-methyl-‘4C-sphingomyelin (Amersham, Buckinghamshire, UK). The substrate was prepared by repeated sonications (up to 30 min) of a mixture of radioactive and nonradioactive sphingomyelin in a ratio of 1:66, suspended in 2% Triton X-100 with 1 mM NaN,. The enzyme assay was carried out in a volume of 0.2 ml, with 0.1 M acetate buffer, pH 5.5, 0.05 mM magnesium acetate, 0.2% Triton X-100,67 nmol of substrate (140,000 cpm). After a 3-h incubation at 37”C, the reaction was stopped with 1 ml of cold 10% (w/v) bovine serum albumin and 0.2 ml of 50% (w/v) trichloracetic acid (TCA). The supernatant solutions, obtained by centrifugation at 1OOOOg for 10 min, were decanted and the precipitates were resuspended in 1 ml of cold 10% TCA and centrifuged. One milliliter of combined supernatants, containing W-labeled reaction products, was mixed with 10 ml of scintillator mixture (Aqualuma) for radioactivity determination. Blanks were between 50-100 cpm. For the ahove enzymes, one unit was defined as micromoles of reaction product generated per hour. The LDH activity was assayed in cuvette with 0.6 ml of 0.1 M sodium phosphate/HCl buffer, pH 7.5, containing 0.27 mM NADH, 0.02% Triton X-100, and sample up to 0.95 ml. The reaction was started with 50 ~1 of 32 mA4 sodium pyruvate. The decrease in absorbance was read at 340 nm with a lin-log recorder. One unit was defined as millimoles of reaction product generated per minute. Experiments with cycloheximide. Epithelial cells (0.5 X 104) were seeded in microtiter plates using as culture medium MEM with 10% FCS, 4 mA4 L-glutamine, 100 IU/ml penicillin, and 100 pg/ml streptomycin. After 24 h, the medium was changed and the cells were conditioned in serum-free MEM for 24 h. Cells were then exposed to
BY
EPITHELIAL
CELLS
195
serum-free MEM without leucine for 6-8 h prior to the addition of 1 FCi of L-[4,5-3H]leucine (Amersham) per well and 5 pg/ml cycloheximide. After 24-h incubation, the media were removed and cells were solubilized with 100 ~1 of 1% SDS. Proteins were then precipitated with 800 ~1 of 10% TCA and filtered on Millipore filters, which were rinsed with 10 ml of 5% TCA and 5 ml of absolute ethanol. The incorporation of [“Hlleucine into proteins was evaluated by scintillation counting. To test the effects of cycloheximide on p-GAM and 0-GIIR activities, 1 X lo6 cells were seeded in Ham’s F-12 medium containing 10% FCS, 4 mA4 L-glutamine, 100 IU/ml penicillin, and 100 pg/ml streptomycin. After 24 h the medium was changed and the cells were conditioned for an additional 24 h in Ham’s F-12 serum-free medium. The cells were then exposed to serum-free medium with or without 5 pg/ ml cycloheximide, incubated for 24 h, and tested for the two hydrolase activities.
RESULTS
Since lysosomal enzyme activities in cell culture may vary according to the growth phase [ 151 and the spreading of cells during attachment on culture dishes may facilitate the exocytosis of lysosomal content [16], the amnion epithelial cells were seeded at nearly confluence density and maintained in culture for 24 h before beginning the experiments. Furthermore, to avoid potential interferences of serum on enzymatic assays, microcultures were established in serum-free conditions. Specific enzyme activities within the cells and in the culture media were referred to cell number (per lo6 cells), because enzyme release may change the total cellular proteins. The number of epithelial cells in microculture was established by measuring the DNA content per well and comparing it with a calibration curve. In 21 experiments the DNA content per cell was estimated to be 15.1 & 0.59 pg (mean -t SEM). The quality of the assays performed was tested with parallel cultures of human fibroblasts, which provided an average value of 10 pg DNA/cell, consistent with data reported in the literature [ 171. To test the potential secretion of lysosomal hydrolases by amnion epithelial cells, we first followed the release of two lysosomal enzyme markers, /3-GAM and P-GUR, for a week at Days 1,4, and 7. At Days 1 and 4, cells which were going to be tesbed at Day 4 and 7, respectively, were exposed to fresh culture medium. As shown in Table 1, the epithelial cells may release about 12% of the total activities of P-GAM and /3-GUR after 24 h in culture, and from about 15 to 17% of total activities during two subsequent periods of 3 days. This process appears to be a true secretion, since concomitantly there is a very low or undetectable release of cytosolic LDH in the extracellular media. The secretion of P-GAM was also evaluated in the absence of EGF and hydrocortisone in the serum-free medium. Up to Day 4, the release of the lysosomal enzyme was virtually identical to the values found above, thereby excluding a stimulatory effect of EGF and/or
196
SCAGGIANTE,
TABLE Secretion glucuronidase
of N-acetyl-fi-glucosaminidase (P-GUR) from Human Enzyme
activity
IN
1 4 7
745 + 121* 680 AZ 118* 697 i 93*
AND
ROMEO
1
@-GUR
Amnion (mU/106
(@-GAM) and @Epithelial Cells
i-
cells) &GUR
$-GAM Days of culture
COMELLI,
95 Secretion 11.0
-t 0.8
14.2
+ 2.0
14.6 k 1.6
IN
5% Secretion
9.8 -+ 0.7* 10.4
+ 0.4*
11.8
t
1.s*
12.6 k 1.6 17.5 * 4.2 16.7 k 2.7
Note. At Days 1 and 4, cells which were going to be tested at Day 4 and 7, respectively, were exposed to fresh culture medium. Results are means + SEM for 6 to 13 experiments. R Secretion was calculated as I OIJT/(OUT + IN) j X 100. The percentage of total LDH activity found extracellularly at the same days of culture were 0.4 k 0.2, 0.5 + 0.5. and I.7 2 1.1 (means k SEM for 8 to 18 determinations). * P > 0.05 (Mann-Witney’s test) for differences among enzyme activities.
1
4
7 DAYS
1
4
$
OF CULTURE
FIG. 1. Extracellular and intracellular N-acetyl-&glucosaminidase (+GAM) and b-glucuronidase (8.GUR) activities in steady state conditions with epithelial cells maintained in culture up to 7 days. The activities are means k SEM for 12 to 14 determinations of fiGAM and for 8 to 12 determinations of 8-GUR.
hydrocortisone on secretion. Thereafter, however, cell survival rapidly diminished as revealed by an increasing recovery of LDH in the medium. Levels of extracellular lysosomal enzymes depend on the rate of secretion as well as on the rate of endocytothe seventh day of culture, the lysosomal enzymes studsis. To study the release of enzymes in a steady-state condition we then maintained the cells in culture for 1 ied may be divided into four classes.The first group with week, without changing the culture medium. Over this the highest rate of secretion (from 19 to 25% of the total includes P-GAM, cr-FUC, and period we did not observe any significant cell damage as enzyme activities) SPHINGO; enzymes of the second group, such as /Jrevealed by dye exclusion tests and by low levels of cytoGUR, (r-MAN, and ARYL, are present in the media at solic LDH in the culture medium (at the seventh day the levels of 10 to 14% of the total activities; the third group extracellular LDH ranged from 2 to 3.5% of the total includes AC PH, LU-GLUC, and Q-GAL, which are recell activity). The results illustrated in Fig. 1 show the release of the two glycosidases into the extracellular medium. For P-GAM, the amount of extracellular enzyme gradually increases to 25% of the total enzyme activity TABLE 2 over a period of 7 days, whereas for fi-GUR the maximal Effect of Cycloheximide on Protein Synthesis and on Nextracellular activity (15% of total) is reached on the acetyl-P-glucosaminidase (fi-GAM) and P-glucuronidase (pGUR) Activities in Cultured Amnion Epithelial Cells (IN) and fourth day. Their Bathing Media (OUT) To establish whether the released enzymes are newly synthesized or are products previously stored in lysoEnzyme activities somes, we then cultured cells in the presence or absence (mU/lO” cells) of 5 pg/ml cycloheximide for 24 h. At this concentration, P-GAM /SGUII [“H]Leucine cycloheximide inhibits by about 85% the incorporation incorporation of [3H]leucine into cellular protein and causes a deIN OUT OUT (cpm) IN crease of about 20% of the intracellular and of 16 to 30% of the extracellular activities of the two lysosomal hy- Control 80037 678 + 61 50.5 2 4.5 21.7 f 5.: 2.7 + 0.5 11440 540 i 50 42.4 -t 5.2 16.8 f 4.7 1.9 t 0.5 drolases (Table 2). However, these differences among Cycloheximide enzyme activities in control and treated samples do not Note. Cells were exposed to serum-free MEM without leucine prior appear to be statistically significant. to the addition of [aH]leucine and cycloheximide (5 fig/ml). After 24 To complement our studies on the appearance of /3- h, media were removed and radioactivity was measured in proteins. In parallel experiments, conducted in GAM and P-GUR in the cell bathing media, we also as- TCA-precipitable F-12 medium and without the addition of [3H]leucine, the sayed other lysosomal enzymes. Figure 2 illustrates the HAM’S activities of &GAM and B-GUR were evaluated in cells and bathing results of some experiments demonstrating a heterogemedia (see Materials and Methods). Results are means +- SEM for neous secretion of lysosomal hydrolases by amnion epi- four experiments. P f 0.05 (Mann-Witney’s test) for differences thelial cells. On the basis of the extracellular activity on among pairs of enzyme activities (controls vs cycloheximide).
LYSOSOMAL 20-
ENZYME
SECRETION
. a-FUC
IO-
t? 2
; nz
20-
o”
, ~-GAL
.a-GAL IAAC PH A a-GLUC
o P-GLUC
IO’
DAYS
OF
CULTURE
FIG. 2. Activities of sphingomyelinase (SPHINGO), cu-fucosidase (n-FUC), ru-mannosidase ( 3): SPHINGO (3), 12.0 i- 0.2; (u-FUC (2), 110.3;
