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Does a fibroblast membrane proteinase affect the binding, uptake and metabolism of epidermal growth factor?
Comp. Biochem. Physiol. Vol. 84B, No. 4, pp. 595 599, 1986 Printed in Great Britain
0305-0491/86 $3.00+ 0.00 Pergamon Journals Ltd
DOES A FIBROBLAST MEMBRANE PROTEINASE AFFECT THE BINDING, UPTAKE A N D METABOLISM OF EPIDERMAL GROWTH FACTOR? G. K. SCOTT and HENG FONG SEOW Department of Biochemistry, University of Auckland, Private Bag, Auckland, New Zealand. (Tel: 737-999) (Received 6 December 1985)
Abstract--1. Pre-treatment of human fibroblasts to inhibit a cell-surfacegrowth-related proteinase inhibits the mitogenic action of epidermal growth factor. 2. It also reduces the binding of epidermal growth factor to these cells, and lowers the rate of internalisation and degradation of the growth factor, but quantitative considerations render it unlikely that these parameters contribute directly to the inhibition of mitogenesis.
INTRODUCTION Epidermal growth factor (EGF) and nerve growth factor (NGF) are both often found in association with proteolytic or esterolytic components, and proteolytic activity is also associated with other, less well-characterised peptide growth factors (Barka, 1980). There is evidence that the proteolytic component catalyses the cleavage of EGF from a larger precursor protein (Frey et al., 1979), but the recentlyreported sequence of the gene for the N G F precursor shows that the proteolytic fl-subunit of N G F does not have the appropriate enzymic specificity to catalyse such a cleavage (Bradshaw, 1983). In the case of EGF, the proteolytic component enhances, though is not essential to, the mitogenic activity of the growth factor itself (Lembach, 1976), and thrombin can also cause a similar enhancement of activity for both EGF (Zetter et al., 1977) and platelet-derived growth factor (PDGF; Zetter and Antoniades, 1979). It has been suggested that proteolytic action may always be necessary for growth factor action (Scott, 1982), and there is now considerable evidence for a ubiquitous cell-surface proteinase which would fulfill this role in the absence of a growth-factor-associated enzyme (Allen et aL, 1981; Allen and Scott, 1983; Pitts and Scott, 1983; Fraser and Scott, 1984a, b; Harper et al., 1984). These experiments depended upon the immunochemical identification or inhibition of the proteinase in cultured cells. Recently the same technique has shown that inhibition of the cell-surface proteinase in human fibroblasts largely inhibits their ability to respond to the mitogenic action of EGF (Scott and Seow, 1985). These latter experiments do not necessarily imply a direct interaction of the proteinase and growth factor in a single mitogenic mechanism; it is equally possible that the observed result could be due to opposite effects on two distinct mechanisms which promote cell growth. As a first step in the further investigation of this problem, we have examined the binding of EGF to human fibroblasts, and its subsequent internalisation and 595
degradation, in the presence and absence of inhibitors of the cell-surface proteinase. MATERIALS AND METHODS
Cell culture and proteinase inhibition Human embryonic lung fibroblasts (strain MP-S) were maintained and cultured .as previously described (Scott and Seow, 1985). The preparation and use of antiproteinase y-globulin (APG) and of ~-l-antitrypsin(a-l-AT) to inhibit cell-surface proteinase, as assayed using phe-val-argnitroanilide as substrate, have also been previously described (Allen et al., 1981; Allen and Scott, 1983; Scott and Seow, 1985). Epidermal growth factor EGF was purchased from Serva (Heidelberg), and a 5 #g/ml stock solution did not hydrolyse the chromogenic peptide substrate, indicating that the growth factor preparation was free from the binding component. It was added to normal growth medium at concentrations of 5 ng/ml for experimentson the relationship between EGF and fibroblast multiplication. For experiments on binding and internalisation, EGF was labelled with ~25iodine, using techniques previously described (Fraser and Scott, 1984a), to a final specifc activity of 1.1 × 104cpm/ng. The stimulation of DNA synthesis by EGF was measured by the acid-insoluble incorporation of [3H]thymidine, as previously described (Scott and Scow, 1985). In the present case, confluent fibroblasts in 15 mm culture-plate wells were incubated in the presence of [3H]thymidine (1 #Ci/ml)~ At intervals after the addition of 5 ng/ml EGF and/ot 300/zg/ml APG the wells were washed three times, and the cells solubilised prior to measurement of radioactivity. EGF binding and internalisation EGF binding and internalisation was initially monitored by treating human fibroblasts in 15mm wells in plastic culture dishes with ~25I-labelled EGF for specified time periods of 15-250 min. At the end of the period, the cells were washed three times with phosphate-buffered saline at 4°C to remove unbound EGF. In the simplest experiments, the cells were then directly solubilised with 0.5 M sodium hydroxide and the total cell-associated radioactivity was measured. In other experiments, cell-surface bound EGF
G . K . SCOTT and HENG FONG SEOW
596
was removed by washing with 0.2 M acetic acid containing 0.5 M sodium chloride (pH 2.5) at 4 C (Haigler et al., 1980). Internalised EGF was then measured by solubilising the cells with 0.5 M sodium hydroxide at 37 for 1 hr. Samples of each solution were used to measure radioactivity. A third type of experiment involved subcellular fractionation by density gradient centrifugation, which was carried out as previously described (Schimmel et al., 1973), as were cytochemical marker enzyme assays (N-acetylglucosaminidase as a lysosomal marker and alkaline phosphatase as a plasma membrane marker: Fraser and Scott, 1984a), All experiments were carried out with cells pre-treated with proteinase inhibitors, in parallel with normal control cells. Plasma membranes from ~25I-EGF-treated fibroblasts were also solubilised in loading solution, and the proteins fractionated by polyacrylamide gel electrophoresis in buffers containing sodium dodecyl sulphate prior to autoradiography (Fraser and Scott, 1984a). The gels used were 6% polyacrylamide rather than 10% as previously described, because of the relatively high molecular weight of the EGF receptor complex. RESULTS
T r e a t m e n t of h u m a n fibroblasts with antiproteinase antibodies results in the inhibition of approximately half of the total cell-surface proteinase activity; the r e m a i n d e r of this activity is inaccessible even to a considerable excess o f the antibodies (Fig.
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Fig. 1. Inhibition of cell-surface proteinase and of multiplication of human fibroblasts. Diploid human fibroblasts between the 6th and 12th passages were grown in Dulbecco's modified minimal Eagle's medium containing 10% foetal calf serum. Aliquots (0.1 ml; 104cells/ml) were dispensed into 96-well plates (for multiplication experiments) or 24-well plates (15 mm; for proteinase assays). After 24 hr, the media were removed and fresh normal or supplemented media added. Supplements were up to 750#g/ml APG (Scott and Seow, 1985) and/or 5 ng/ml EGF. In multiplication experiments, the media were changed, and the cells counted, daily. The results are expressed as the percentage of cell counts in control cultures after 96 hr. The proteinase assays were carried out over 6 hr on the second day of the experiment. The release ofp-nitrophenol from the peptidechromogen was measured at 405 nm, and expressed as a percentage of values for control cultures. O - - O proteinase activity; • • cell multiplication; A - - - - A cell multiplication in the presence of EGF.
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Fig. 2. Effect of antiproteinase 7-globulin on the stimulation of DNA synthesis by epidermal growth factor. Human fibroblasts were grown in 96-well plates for 24hr (ca. 2 x 103 cells/well) before the addition of APG (75 llg/ml) and/or EGF (5 ng/ml). [3H]thymidine (1 l~Ci/ml) was added immediately to one series of welts, and after 3, 10, 16, 20 and 21 hr to other series. After a 2 hr exposure to [3H]thymidine, each series of wells was washed three times with serum-free medium, and the cells released by trypsin treatment, collected on 0.2,urn filters, washed three times with 10% trichloracetic acid and dried prior to scintillation counting. O O control; Q • APG: • - A EGF: A--A EGF + APG.
1). This effect is roughly paralleled by the total inhibition of cell multiplication by the same antibodies. Cell multiplication is also totally inhibited in the presence of E G F , t h o u g h at rather higher antibody concentrations. These results confirm similar earlier findings (Allen et al., 1981; Scott and Scow, 1985), a n d are consistent with the inhibition of D N A synthesis (Fig. 2). Figure 3 summarises the results of E G F binding studies. It is clear that the E G F - b i n d i n g capacity of h u m a n fibroblasts is more readily saturated when they have been previously treated with a n t i p r o t e i n a s e antibodies. However, in the range o f E G F concentrations which are used to stimulate cell multiplication, there is no significant difference in E G F binding between n o r m a l and A P G - t r e a t e d cells. H u m a n ~ - l - a n t i t r y p s i n has a similar effect to A P G : we have previously s h o w n t h a t this protein proteinase inhibitor acts on the growth-related proteinase in the same way as A P G (Scott a n d Seow, 1985). A preliminary study of E G F internalisation is presented in Fig. 4. Following t r e a t m e n t with ~2sI-EGF, growth factor b o u n d to the cell surface was r e m o v e d by washing the cells with 0.2 M acetic a c i d / 0 . h M sodium chloride. The cells were then solubilised in 0.5 M sodium hydroxide. The radioactivity in each fraction was t a k e n as a measure of cell-bound a n d internalised E G F , respectively. In n o r m a l cells, the E G F b o u n d at the surface declines more rapidly over the course of the experiment, and the intracellular E G F levels are at a relatively high level t h r o u g h o u t . In contrast, the A P G - t r e a t e d cells display a less rapid decline in cell-bound E G F , coupled with initially low levels of internalised E G F which steadily rise d u r i n g the experiment.
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EGF added (ng/ml) Fig. 3. Effect of proteinase inhibitors on the binding of epidermal growth factor to human fibroblasts. Fibroblasts (ca. 2 x 104cells in 15ram wells) were pretreated with antiproteinase 7-globulin (150/~g/ml) or e-l-antitrypsin (25 ,ug/ml) for 24 hr and then incubated with 125I-EGFfor 1 hr at 37°C. The cells were washed three times with phosphate-buffered saline at 4°C and solubilised with 0.5 M NaOH for 1 hr at 37° prior to y-counting for radioactivity. © © control; • • APG; /~ A or-I-AT.
597
to bind, internalise and degrade epidermal growth factor. In particular, the cells become saturated with E G F at a markedly lower concentration. This result could imply lower numbers of E G F receptors, or a lower EGF-receptor affinity, or less rapid internalisation of EGF-receptor complexes. However, the binding of E G F within the physiologically-relevant concentration range (i.e. up to 50ng/ml) is not greatly altered. In normal fibroblasts, bound 125I-EGF is initially concentrated at the cell surface but is rapidly internalised to the lysosomal fraction. This result is comparable with reports by other workers. The radioactivity is ultimately found in the cytoplasmic fraction, which has been explained in terms of the lysosomal degradation of E G F and secretion of the low-molecular weight products (Carpenter and Cohen, 1976). The same pathway is observed in proteinase-inhibited cells, but at a considerably reduced rate relative to normal cells. Under the experimental conditions used, i.e. with the pre-incubation of cells with A P G prior to the administration of EGF, it is likely that this result simply reflects the lower metabolic activity of cells treated with APG. In any event, the increased time required for inter-
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These data strongly suggest that E G F internalisation is slower in APG-treated than in normal fibroblasts. This conclusion is supported by subcellular fractionation of fibroblasts during the course of E G F internalisation (Fig. 5). One hour after the administration of EGF, the associated radioisotope is predominantly in the lysosomal fraction of normal cells, although there is a small amount at the top of the gradient, which we have taken to represent degradation products of the labelled growth factor which have been secreted from the cells. In APGtreated fibroblasts, this totally-degraded component is absent, and the intracellular radioisotope is equally distributed between lysosomal and plasma membrane components. Two hours after E G F administration, the associated radioisotope is equally distributed between lysosomal and secreted compartments in control cells, whereas in APG-treated cells, the majority of the isotope is still intracellular and some of it is still associated with the plasma membrane fraction. One possible effect of an active cell-surface proteinase would be on the E G F receptor, or on the EGF-receptor complex. We have examined the EGFreceptor complex by autoradiography of fibroblast membrane proteins separated by electrophoresis following the exposure of the cells to 125I-EGF. The apparent molecular weight of the EGF-receptor complex is similar in both normal and APG-treated cells (Fig. 6). DISCUSSION
When human fibroblasts are treated with reagents which inhibit their growth-related cell-surface proteinase, there is also a marked effect on their ability
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Fig. 4. Effect of antiproteinase 7-globulin on the rate of internalisation of epidermal growth factor. Cells were grown and treated with APG as previously described (Figs 1 and 2), and incubated with 12~I-EGF(5 ng/ml) for specified times followed by three washes with phosphate-buffered saline at 4°. Cell-surface-bound EGF was removed by washing with 0.2 M acetic acid/0.5 M sodium chloride pH 2.5, and internalised EGF was measured following cell solubilisation with 0.5 M sodium hydroxide. Radioactivity is expressed as a percentage of total 125I-EGFadded (5/t Ci). Hatched bars in the histogram represent normal cells and open bars represent APG-treated cells. (a) Cell-surface-bound EGF; (b) internalised EGF.
G . K . SCOTTand HENGFONG SEOW
598
nalisation of E G F in APG-treated cells is still much less than the time delay between the administration of E G F and the observation of increased D N A synthesis (Fig. 2). It thus seems unlikely that the lower rate of E G F internalisation and degradation is directly linked to the inhibition of mitogenesis by APG. An obvious way in which an extracellular proteinase could influence E G F uptake would be by modification or degradation of the E G F receptor. However, there is no evidence for a change in apparent molecular weight of the E G F receptor in APGtreated fibroblasts, although the experiment does not exclude the possibility of proteolytic modification which does not result in a fall in molecular weight. A lower-molecular-weight species of E G F receptor has been reported by other workers, but is apparently due to artefactual degradation by a cytoplasmic thiol proteinase during cell fractionation (Cohen et al.. 1982). Although we have observed that inhibition of the growth-related proteinase does result in a reduction in the extent of E G F binding to human fibroblasts,
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7 4 6 8 10 12 Fraction no. (2 ml) Fig. 5. Subcellular distribution of '251-EGF-receptor complex and degradation products. (a) Separation of organelles by density-gradient centrifugation and assay of marker enzymes, measured by release ofp-nitrophenol from appropriate substrates O - - - © refractive index of sucrose density gradient; 0 - - 0 plasma membrane (alkaline phosphatase); A - - A lysosomes (N-acetylglucosaminidase). (b) Cells were treated with ]2SI-EGF for 1 hr and washed as described in the legends to Figs 3 and 4, and then lysed and fractionated. © - - © control; 0 - - 0 APG-treated cells. (c) As for (b), but cells exposed to ~251-EGFfor 2 hr before fractionation.
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Fig. 6. Electrophoresis of EGF-receptor complex from normal and APG-treated fibroblasts. Cells were treated with t25I-EGF and washed as described in the legends to Figs 3 and 4, and then solubilised prior to electrophoresis on 6% polyacrylamide gels in the presence of sodium dodecyl sulphate. Marker proteins of known molecular weight (myosin 200 K; 7-globulin 150 K; phosphorylase b 98 K and transferrin 80 K) were used to calibrate the Coomassie Blue-stained gels, which were then dried and overlaid with Kodak X-Omat film. After 2 months, a developed film was scanned with a Helena Quickscan densitometer. (a) normal cells; (b) APG-treated cells.
and also in a reduced rate of E G F internalisation and degradation, it is unlikely that either of these phenomena are directly connected with the inhibition of mitogenesis. On the available evidence, it seems more likely that the "antimitogenic" step is located in some other biochemical mechanism which exerts an indirect influence on EGF-stimulated mitogenesis, or that it is in the intracellular pathway which mediates the action of EGF. This pathway includes the tyrosinespecific kinase activity which is associated with the E G F receptor (Cohen et al., 1982), and the phosphatidylinositol second messenger system which is subsequently activated (Berridge, 1984). It would seem worthwhile to examine the effects of inhibition of the growth-related proteinase on the operation of these systems. Acknowledgements--We wish to thank the Medical Re-
search Council of New Zealand and the Auckland University Research Grants Committee for financial support, and Mrs. M. Mackie, who typed the manuscript.
REFERENCES
Allen R. J., Rattray S. and Scott, G. K. (1981) Preliminary evidence that thrombin may mimic a naturally-occurring proteinase in cultured cells. Biosci. Rep. 1, 881 884.
Fibroblast membrane proteinase Allen R. J. and Scott G. K. (1983) A neutral proteinase from human leukocyte membranes. Int. J. Biochem. 15, 151 154. Barka T. (1980) Biologically-active peptides in submandibular glands. J. Histochem. Cytochem. 28, 836-859. Berridge M. (1984) Inositol trisphosphate and diacylglycerol as second messengers. Biochem. J. 220, 345-360. Bradshaw R. (1983) What cloned genes can tell us about nerve growth factor. Nature (Lond.) 303, 715. Carpenter G. and Cohen S. (1976) 125I-Labeled human epidermal growth factor: binding, internalisation and degradation in human fibroblasts. J. Cell Biol. 71, 159-171. Cohen S., Ushiro H., Stoscheck C. and Chinkers M. (1982) A native 170,000 epidermal growth factor receptor-kinase complex from shed plasma membrane vesicles. J. biol. Chem. 257, 1523-1531. Fraser J. D., and Scott G. K. (1984a) Mitogenic proteinases from human leukocytes. Molecular Immunol. 21, 311-320. Fraser J. D. and Scott G. K. (1984b) Membrane proteinases from normal and neoplastic tissues in man and the rat. Comp. Biochem. Biophys. 7913, 105-111. Frey P., Forand R., Maciag T. and Shooter E. M. (1979) The biosynthetic precursor of epidermal growth factor and the mechanism of its processing. Proc. Natl Acad. Sci. USA 76, 6294-6298. Haigler H. T., Maxfield F. R., Willingham M. C. and Pastan, I. (1980) Dansyl-cadaverine inhibits internalisation of ~25I-epidermal growth factor in BALB 3T3 cells. J. biol. Chem. 255, 1239-1241.
C.B.P. 84/4[~-L
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Harper L., Scott G. K. and Seow H. F. (1984) Antibody affinity chromatography of human proteinases and related proteins. Comp. Biochem. Physiol. 7gB, 231-235. Lembach K. J. (1976) Induction of human fibroblast proliferation by epidermal growth factor (EGF): enhancement by an EGF-binding arglnine esterase and by ascorbate. Proc. Natl Acad. Sci. USA 73, 183-187. Pitts J. D. and Scott G. K. (1983) Growth inhibition of normal, tumour and transformed cells by antibodies to a cell-surface proteinase. Biosci. Rep. 3, 47-51. Schimmel S. D., Kent C., Bischoff R. and Vagelos P. R. (1973) Plasma membranes from cultured muscle cells: isolation procedure and separation of putative plasmamembrane marker enzymes. Proc. Natl Acad. Sci. USA 70, 3195-3199. Scott G. K. (1982) Peptide growth factors and mitogenic proteinases. Med. Hypotheses 9, 307-310. Scott G. K. and Seow H. F. (1985) Further evidence for a cell surface proteinase essential to the growth of cultured fibroblasts. Exp. Cell Res. 158, 41-52. Zetter B. R. and Antoniades H. N. (1979) Stimulation of human vascular endothelial cell growth by a plateletderived growth factor and thrombin. J. Supramol. Struct. I1, 361-370. Zetter B. R., Sun T. T., Chen L. B. and Buchanan J. M. (1977) Thrombin potentiates the mitogenic response of cultured fibroblasts to serum and other growthpromoting agents. J. cell. Physiol. 92, 233-239.
0305-0491/86 $3.00+ 0.00 Pergamon Journals Ltd
DOES A FIBROBLAST MEMBRANE PROTEINASE AFFECT THE BINDING, UPTAKE A N D METABOLISM OF EPIDERMAL GROWTH FACTOR? G. K. SCOTT and HENG FONG SEOW Department of Biochemistry, University of Auckland, Private Bag, Auckland, New Zealand. (Tel: 737-999) (Received 6 December 1985)
Abstract--1. Pre-treatment of human fibroblasts to inhibit a cell-surfacegrowth-related proteinase inhibits the mitogenic action of epidermal growth factor. 2. It also reduces the binding of epidermal growth factor to these cells, and lowers the rate of internalisation and degradation of the growth factor, but quantitative considerations render it unlikely that these parameters contribute directly to the inhibition of mitogenesis.
INTRODUCTION Epidermal growth factor (EGF) and nerve growth factor (NGF) are both often found in association with proteolytic or esterolytic components, and proteolytic activity is also associated with other, less well-characterised peptide growth factors (Barka, 1980). There is evidence that the proteolytic component catalyses the cleavage of EGF from a larger precursor protein (Frey et al., 1979), but the recentlyreported sequence of the gene for the N G F precursor shows that the proteolytic fl-subunit of N G F does not have the appropriate enzymic specificity to catalyse such a cleavage (Bradshaw, 1983). In the case of EGF, the proteolytic component enhances, though is not essential to, the mitogenic activity of the growth factor itself (Lembach, 1976), and thrombin can also cause a similar enhancement of activity for both EGF (Zetter et al., 1977) and platelet-derived growth factor (PDGF; Zetter and Antoniades, 1979). It has been suggested that proteolytic action may always be necessary for growth factor action (Scott, 1982), and there is now considerable evidence for a ubiquitous cell-surface proteinase which would fulfill this role in the absence of a growth-factor-associated enzyme (Allen et aL, 1981; Allen and Scott, 1983; Pitts and Scott, 1983; Fraser and Scott, 1984a, b; Harper et al., 1984). These experiments depended upon the immunochemical identification or inhibition of the proteinase in cultured cells. Recently the same technique has shown that inhibition of the cell-surface proteinase in human fibroblasts largely inhibits their ability to respond to the mitogenic action of EGF (Scott and Seow, 1985). These latter experiments do not necessarily imply a direct interaction of the proteinase and growth factor in a single mitogenic mechanism; it is equally possible that the observed result could be due to opposite effects on two distinct mechanisms which promote cell growth. As a first step in the further investigation of this problem, we have examined the binding of EGF to human fibroblasts, and its subsequent internalisation and 595
degradation, in the presence and absence of inhibitors of the cell-surface proteinase. MATERIALS AND METHODS
Cell culture and proteinase inhibition Human embryonic lung fibroblasts (strain MP-S) were maintained and cultured .as previously described (Scott and Seow, 1985). The preparation and use of antiproteinase y-globulin (APG) and of ~-l-antitrypsin(a-l-AT) to inhibit cell-surface proteinase, as assayed using phe-val-argnitroanilide as substrate, have also been previously described (Allen et al., 1981; Allen and Scott, 1983; Scott and Seow, 1985). Epidermal growth factor EGF was purchased from Serva (Heidelberg), and a 5 #g/ml stock solution did not hydrolyse the chromogenic peptide substrate, indicating that the growth factor preparation was free from the binding component. It was added to normal growth medium at concentrations of 5 ng/ml for experimentson the relationship between EGF and fibroblast multiplication. For experiments on binding and internalisation, EGF was labelled with ~25iodine, using techniques previously described (Fraser and Scott, 1984a), to a final specifc activity of 1.1 × 104cpm/ng. The stimulation of DNA synthesis by EGF was measured by the acid-insoluble incorporation of [3H]thymidine, as previously described (Scott and Scow, 1985). In the present case, confluent fibroblasts in 15 mm culture-plate wells were incubated in the presence of [3H]thymidine (1 #Ci/ml)~ At intervals after the addition of 5 ng/ml EGF and/ot 300/zg/ml APG the wells were washed three times, and the cells solubilised prior to measurement of radioactivity. EGF binding and internalisation EGF binding and internalisation was initially monitored by treating human fibroblasts in 15mm wells in plastic culture dishes with ~25I-labelled EGF for specified time periods of 15-250 min. At the end of the period, the cells were washed three times with phosphate-buffered saline at 4°C to remove unbound EGF. In the simplest experiments, the cells were then directly solubilised with 0.5 M sodium hydroxide and the total cell-associated radioactivity was measured. In other experiments, cell-surface bound EGF
G . K . SCOTT and HENG FONG SEOW
596
was removed by washing with 0.2 M acetic acid containing 0.5 M sodium chloride (pH 2.5) at 4 C (Haigler et al., 1980). Internalised EGF was then measured by solubilising the cells with 0.5 M sodium hydroxide at 37 for 1 hr. Samples of each solution were used to measure radioactivity. A third type of experiment involved subcellular fractionation by density gradient centrifugation, which was carried out as previously described (Schimmel et al., 1973), as were cytochemical marker enzyme assays (N-acetylglucosaminidase as a lysosomal marker and alkaline phosphatase as a plasma membrane marker: Fraser and Scott, 1984a), All experiments were carried out with cells pre-treated with proteinase inhibitors, in parallel with normal control cells. Plasma membranes from ~25I-EGF-treated fibroblasts were also solubilised in loading solution, and the proteins fractionated by polyacrylamide gel electrophoresis in buffers containing sodium dodecyl sulphate prior to autoradiography (Fraser and Scott, 1984a). The gels used were 6% polyacrylamide rather than 10% as previously described, because of the relatively high molecular weight of the EGF receptor complex. RESULTS
T r e a t m e n t of h u m a n fibroblasts with antiproteinase antibodies results in the inhibition of approximately half of the total cell-surface proteinase activity; the r e m a i n d e r of this activity is inaccessible even to a considerable excess o f the antibodies (Fig.
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Fig. 1. Inhibition of cell-surface proteinase and of multiplication of human fibroblasts. Diploid human fibroblasts between the 6th and 12th passages were grown in Dulbecco's modified minimal Eagle's medium containing 10% foetal calf serum. Aliquots (0.1 ml; 104cells/ml) were dispensed into 96-well plates (for multiplication experiments) or 24-well plates (15 mm; for proteinase assays). After 24 hr, the media were removed and fresh normal or supplemented media added. Supplements were up to 750#g/ml APG (Scott and Seow, 1985) and/or 5 ng/ml EGF. In multiplication experiments, the media were changed, and the cells counted, daily. The results are expressed as the percentage of cell counts in control cultures after 96 hr. The proteinase assays were carried out over 6 hr on the second day of the experiment. The release ofp-nitrophenol from the peptidechromogen was measured at 405 nm, and expressed as a percentage of values for control cultures. O - - O proteinase activity; • • cell multiplication; A - - - - A cell multiplication in the presence of EGF.
10
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5
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i
;
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Time (hr)
Fig. 2. Effect of antiproteinase 7-globulin on the stimulation of DNA synthesis by epidermal growth factor. Human fibroblasts were grown in 96-well plates for 24hr (ca. 2 x 103 cells/well) before the addition of APG (75 llg/ml) and/or EGF (5 ng/ml). [3H]thymidine (1 l~Ci/ml) was added immediately to one series of welts, and after 3, 10, 16, 20 and 21 hr to other series. After a 2 hr exposure to [3H]thymidine, each series of wells was washed three times with serum-free medium, and the cells released by trypsin treatment, collected on 0.2,urn filters, washed three times with 10% trichloracetic acid and dried prior to scintillation counting. O O control; Q • APG: • - A EGF: A--A EGF + APG.
1). This effect is roughly paralleled by the total inhibition of cell multiplication by the same antibodies. Cell multiplication is also totally inhibited in the presence of E G F , t h o u g h at rather higher antibody concentrations. These results confirm similar earlier findings (Allen et al., 1981; Scott and Scow, 1985), a n d are consistent with the inhibition of D N A synthesis (Fig. 2). Figure 3 summarises the results of E G F binding studies. It is clear that the E G F - b i n d i n g capacity of h u m a n fibroblasts is more readily saturated when they have been previously treated with a n t i p r o t e i n a s e antibodies. However, in the range o f E G F concentrations which are used to stimulate cell multiplication, there is no significant difference in E G F binding between n o r m a l and A P G - t r e a t e d cells. H u m a n ~ - l - a n t i t r y p s i n has a similar effect to A P G : we have previously s h o w n t h a t this protein proteinase inhibitor acts on the growth-related proteinase in the same way as A P G (Scott a n d Seow, 1985). A preliminary study of E G F internalisation is presented in Fig. 4. Following t r e a t m e n t with ~2sI-EGF, growth factor b o u n d to the cell surface was r e m o v e d by washing the cells with 0.2 M acetic a c i d / 0 . h M sodium chloride. The cells were then solubilised in 0.5 M sodium hydroxide. The radioactivity in each fraction was t a k e n as a measure of cell-bound a n d internalised E G F , respectively. In n o r m a l cells, the E G F b o u n d at the surface declines more rapidly over the course of the experiment, and the intracellular E G F levels are at a relatively high level t h r o u g h o u t . In contrast, the A P G - t r e a t e d cells display a less rapid decline in cell-bound E G F , coupled with initially low levels of internalised E G F which steadily rise d u r i n g the experiment.
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EGF added (ng/ml) Fig. 3. Effect of proteinase inhibitors on the binding of epidermal growth factor to human fibroblasts. Fibroblasts (ca. 2 x 104cells in 15ram wells) were pretreated with antiproteinase 7-globulin (150/~g/ml) or e-l-antitrypsin (25 ,ug/ml) for 24 hr and then incubated with 125I-EGFfor 1 hr at 37°C. The cells were washed three times with phosphate-buffered saline at 4°C and solubilised with 0.5 M NaOH for 1 hr at 37° prior to y-counting for radioactivity. © © control; • • APG; /~ A or-I-AT.
597
to bind, internalise and degrade epidermal growth factor. In particular, the cells become saturated with E G F at a markedly lower concentration. This result could imply lower numbers of E G F receptors, or a lower EGF-receptor affinity, or less rapid internalisation of EGF-receptor complexes. However, the binding of E G F within the physiologically-relevant concentration range (i.e. up to 50ng/ml) is not greatly altered. In normal fibroblasts, bound 125I-EGF is initially concentrated at the cell surface but is rapidly internalised to the lysosomal fraction. This result is comparable with reports by other workers. The radioactivity is ultimately found in the cytoplasmic fraction, which has been explained in terms of the lysosomal degradation of E G F and secretion of the low-molecular weight products (Carpenter and Cohen, 1976). The same pathway is observed in proteinase-inhibited cells, but at a considerably reduced rate relative to normal cells. Under the experimental conditions used, i.e. with the pre-incubation of cells with A P G prior to the administration of EGF, it is likely that this result simply reflects the lower metabolic activity of cells treated with APG. In any event, the increased time required for inter-
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These data strongly suggest that E G F internalisation is slower in APG-treated than in normal fibroblasts. This conclusion is supported by subcellular fractionation of fibroblasts during the course of E G F internalisation (Fig. 5). One hour after the administration of EGF, the associated radioisotope is predominantly in the lysosomal fraction of normal cells, although there is a small amount at the top of the gradient, which we have taken to represent degradation products of the labelled growth factor which have been secreted from the cells. In APGtreated fibroblasts, this totally-degraded component is absent, and the intracellular radioisotope is equally distributed between lysosomal and plasma membrane components. Two hours after E G F administration, the associated radioisotope is equally distributed between lysosomal and secreted compartments in control cells, whereas in APG-treated cells, the majority of the isotope is still intracellular and some of it is still associated with the plasma membrane fraction. One possible effect of an active cell-surface proteinase would be on the E G F receptor, or on the EGF-receptor complex. We have examined the EGFreceptor complex by autoradiography of fibroblast membrane proteins separated by electrophoresis following the exposure of the cells to 125I-EGF. The apparent molecular weight of the EGF-receptor complex is similar in both normal and APG-treated cells (Fig. 6). DISCUSSION
When human fibroblasts are treated with reagents which inhibit their growth-related cell-surface proteinase, there is also a marked effect on their ability
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Fig. 4. Effect of antiproteinase 7-globulin on the rate of internalisation of epidermal growth factor. Cells were grown and treated with APG as previously described (Figs 1 and 2), and incubated with 12~I-EGF(5 ng/ml) for specified times followed by three washes with phosphate-buffered saline at 4°. Cell-surface-bound EGF was removed by washing with 0.2 M acetic acid/0.5 M sodium chloride pH 2.5, and internalised EGF was measured following cell solubilisation with 0.5 M sodium hydroxide. Radioactivity is expressed as a percentage of total 125I-EGFadded (5/t Ci). Hatched bars in the histogram represent normal cells and open bars represent APG-treated cells. (a) Cell-surface-bound EGF; (b) internalised EGF.
G . K . SCOTTand HENGFONG SEOW
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nalisation of E G F in APG-treated cells is still much less than the time delay between the administration of E G F and the observation of increased D N A synthesis (Fig. 2). It thus seems unlikely that the lower rate of E G F internalisation and degradation is directly linked to the inhibition of mitogenesis by APG. An obvious way in which an extracellular proteinase could influence E G F uptake would be by modification or degradation of the E G F receptor. However, there is no evidence for a change in apparent molecular weight of the E G F receptor in APGtreated fibroblasts, although the experiment does not exclude the possibility of proteolytic modification which does not result in a fall in molecular weight. A lower-molecular-weight species of E G F receptor has been reported by other workers, but is apparently due to artefactual degradation by a cytoplasmic thiol proteinase during cell fractionation (Cohen et al.. 1982). Although we have observed that inhibition of the growth-related proteinase does result in a reduction in the extent of E G F binding to human fibroblasts,
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7 4 6 8 10 12 Fraction no. (2 ml) Fig. 5. Subcellular distribution of '251-EGF-receptor complex and degradation products. (a) Separation of organelles by density-gradient centrifugation and assay of marker enzymes, measured by release ofp-nitrophenol from appropriate substrates O - - - © refractive index of sucrose density gradient; 0 - - 0 plasma membrane (alkaline phosphatase); A - - A lysosomes (N-acetylglucosaminidase). (b) Cells were treated with ]2SI-EGF for 1 hr and washed as described in the legends to Figs 3 and 4, and then lysed and fractionated. © - - © control; 0 - - 0 APG-treated cells. (c) As for (b), but cells exposed to ~251-EGFfor 2 hr before fractionation.
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Fig. 6. Electrophoresis of EGF-receptor complex from normal and APG-treated fibroblasts. Cells were treated with t25I-EGF and washed as described in the legends to Figs 3 and 4, and then solubilised prior to electrophoresis on 6% polyacrylamide gels in the presence of sodium dodecyl sulphate. Marker proteins of known molecular weight (myosin 200 K; 7-globulin 150 K; phosphorylase b 98 K and transferrin 80 K) were used to calibrate the Coomassie Blue-stained gels, which were then dried and overlaid with Kodak X-Omat film. After 2 months, a developed film was scanned with a Helena Quickscan densitometer. (a) normal cells; (b) APG-treated cells.
and also in a reduced rate of E G F internalisation and degradation, it is unlikely that either of these phenomena are directly connected with the inhibition of mitogenesis. On the available evidence, it seems more likely that the "antimitogenic" step is located in some other biochemical mechanism which exerts an indirect influence on EGF-stimulated mitogenesis, or that it is in the intracellular pathway which mediates the action of EGF. This pathway includes the tyrosinespecific kinase activity which is associated with the E G F receptor (Cohen et al., 1982), and the phosphatidylinositol second messenger system which is subsequently activated (Berridge, 1984). It would seem worthwhile to examine the effects of inhibition of the growth-related proteinase on the operation of these systems. Acknowledgements--We wish to thank the Medical Re-
search Council of New Zealand and the Auckland University Research Grants Committee for financial support, and Mrs. M. Mackie, who typed the manuscript.
REFERENCES
Allen R. J., Rattray S. and Scott, G. K. (1981) Preliminary evidence that thrombin may mimic a naturally-occurring proteinase in cultured cells. Biosci. Rep. 1, 881 884.
Fibroblast membrane proteinase Allen R. J. and Scott G. K. (1983) A neutral proteinase from human leukocyte membranes. Int. J. Biochem. 15, 151 154. Barka T. (1980) Biologically-active peptides in submandibular glands. J. Histochem. Cytochem. 28, 836-859. Berridge M. (1984) Inositol trisphosphate and diacylglycerol as second messengers. Biochem. J. 220, 345-360. Bradshaw R. (1983) What cloned genes can tell us about nerve growth factor. Nature (Lond.) 303, 715. Carpenter G. and Cohen S. (1976) 125I-Labeled human epidermal growth factor: binding, internalisation and degradation in human fibroblasts. J. Cell Biol. 71, 159-171. Cohen S., Ushiro H., Stoscheck C. and Chinkers M. (1982) A native 170,000 epidermal growth factor receptor-kinase complex from shed plasma membrane vesicles. J. biol. Chem. 257, 1523-1531. Fraser J. D., and Scott G. K. (1984a) Mitogenic proteinases from human leukocytes. Molecular Immunol. 21, 311-320. Fraser J. D. and Scott G. K. (1984b) Membrane proteinases from normal and neoplastic tissues in man and the rat. Comp. Biochem. Biophys. 7913, 105-111. Frey P., Forand R., Maciag T. and Shooter E. M. (1979) The biosynthetic precursor of epidermal growth factor and the mechanism of its processing. Proc. Natl Acad. Sci. USA 76, 6294-6298. Haigler H. T., Maxfield F. R., Willingham M. C. and Pastan, I. (1980) Dansyl-cadaverine inhibits internalisation of ~25I-epidermal growth factor in BALB 3T3 cells. J. biol. Chem. 255, 1239-1241.
C.B.P. 84/4[~-L
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Harper L., Scott G. K. and Seow H. F. (1984) Antibody affinity chromatography of human proteinases and related proteins. Comp. Biochem. Physiol. 7gB, 231-235. Lembach K. J. (1976) Induction of human fibroblast proliferation by epidermal growth factor (EGF): enhancement by an EGF-binding arglnine esterase and by ascorbate. Proc. Natl Acad. Sci. USA 73, 183-187. Pitts J. D. and Scott G. K. (1983) Growth inhibition of normal, tumour and transformed cells by antibodies to a cell-surface proteinase. Biosci. Rep. 3, 47-51. Schimmel S. D., Kent C., Bischoff R. and Vagelos P. R. (1973) Plasma membranes from cultured muscle cells: isolation procedure and separation of putative plasmamembrane marker enzymes. Proc. Natl Acad. Sci. USA 70, 3195-3199. Scott G. K. (1982) Peptide growth factors and mitogenic proteinases. Med. Hypotheses 9, 307-310. Scott G. K. and Seow H. F. (1985) Further evidence for a cell surface proteinase essential to the growth of cultured fibroblasts. Exp. Cell Res. 158, 41-52. Zetter B. R. and Antoniades H. N. (1979) Stimulation of human vascular endothelial cell growth by a plateletderived growth factor and thrombin. J. Supramol. Struct. I1, 361-370. Zetter B. R., Sun T. T., Chen L. B. and Buchanan J. M. (1977) Thrombin potentiates the mitogenic response of cultured fibroblasts to serum and other growthpromoting agents. J. cell. Physiol. 92, 233-239.