Journal of Neuroscience Research 31: 14-20 (1992)
Receptor-Mediated Endocytosis and Degradation of Insulin-Like Growth Factor I and 11 in Neonatal Rat Astrocytes M. Auletta, F.C. Nielsen, and S. Gammeltoft Department of Clinical Chemistry, Bispebjerg Hospital, Copenhagen, Denmark Lund et al., 1986; Saraet al., 1986; Rotwein et al., 1988; Stylianopoulou et al., 1988). Two types of IGF receptors are distributed throughout the fetal and adult mammalian CNS (Sara et al., 1983; Gammeltoft et al., 1985). Recent studies have demonstrated that astrocytes have receptors for IGF-I and IGF-I1 and that IGFs stimulate DNA synthesis via activation of the IGF-I receptor tyrosine kinase (Ballotti et al., 1987; Han et al., 1987; Shemer et al., 1987). On the basis of the finding that neonatal rat astrocytes express both IGF-I and IGF-I1 (Ballotti et al., 1987; Rotwein et al., 1988), it has been suggested that IGFs stimulate the clonal expansion of astrocytes in neonatal rat brain by an autocrine or paracrine mechanism (Gammeltoft et al., 1988). This raises the question of whether IGF-I and IGF-11, which are produced by astrocytes and interact with cell-surface receptors, are also removed and processed by these cells. Receptor-mediated endocytosis of polypeptide hormones, growth factors, and neuropeptides represents the major pathway in the breakdown of bioactive substances (Gammeltoft, 1991). In various cell types, IGF-I and IGF-I1 are internalized and degraded by interaction with IGF-I receptors and mannose 6-phosphate/IGF-I1 (Man6-P/IGF-11) receptors, respectively (Bar et al., 1986; Banskota et al., 1986; Frank et al., 1986; Hachiya et al., 1986; Oka et al., 1985; Schalch et al., 1985; Nielsen et al., 1991. In the present study, we show that IGF-I and IGF-I1 are processed by two distinct pathways of receptor-meKey words: insulin-like growth factor, IGF-I recep- diated endocytosis and degradation in neonatal rat astrotor, IGF-I1 receptor, Man-6-P receptor, endocytosis, cytes. Furthermore, the expression of IGF-I receptors IGF degradation, central nervous system, glial cells and Man-6-P/IGF-I1 receptors on the cell surface seems to be regulated by independent mechanisms. INTRODUCTION
Receptor-mediated internalization and degradation of insulin-like growth factors, IGF-I and IGF-11, were studied in primary cultures of neonatal rat astrocytes. Surface-bound IGF-I1 was rapidly internalize& and 80% of cell-associated radioactivity was located intracellularly after 30 min. IGF-I was internalized at a slower rate, and only 40% of cell-associated radioactivity was inside the cell after 30 min. A pulse-chase experiment demonstrated that 55% and 70% of internalized IGF-I and IGF-11, respectively, was degraded to free amino acids after a 3-hr chase. Lysosomal and protease inhibitors had different effects on the binding, internalization, and processing of IGF-I and IGF-11. Inhibition of lysosomal acidification by chloroquine increased the amounts of surface-bound IGF-I1 and intracellular IGF-I and reduced the degradation of IGF-I. The chelating agent phenanthroline increased the surface binding of IGFI and IGF-I1 and internalization of IGF-I1 and reduced the degradation of IGF-I and IGF-11. Finally, receptor-bound IGF-I1 on the cell surface was decreased with increasing cell density, whereas IGF-I binding was unaltered. Our data suggest that cellsurface expression of IGF-I receptors and IGF-I1 receptors is regulated by different mechanisms and that receptor-bound IGF-I and IGF-I1 are trafficked and processed by different intracellular pathways in neonatal rat astrocytes.
Received January 18, 1991; accepted May 29, 1991.
Insulin-like growth factors (IGF) I and 11 are polypeptides structurally similar to insulin that act as mitogens in various fetal and adult tissues (Froesch et al., 1985; Gammeltoft, 1989). In the central nervous system (CNS) of fetal and adult humans and rat, gene and protein expression of IGF-I and IGF-I1 has been demonstrated (Haselbacher et al., 1985; Brown et al., 1986; 0 1992 Wiley-Liss, Inc.
Address reprint requests to Dr. Steen Gammeltoft, Department of Clinical Chemistry, Bispebjerg Hospital, DK-2400 Copenhagen NV, Denmark. M. Auletta is presently at Fourth Division of Internal Medicine, Second Faculty of Medicine and Surgery, University of Napoli, Italy. F.C. Nielsen is presently at the Department of Clinical Chemistry, Rigshospitalet, University of Copenhagen, Denmark.
Endocytosis of IGF-I and IGF-I1 in Astrocytes MATERIALS AND METHODS Materials Recombinant human IGF-I and IGF-I1 were purchased from Boehringer (Mannheim, West Germany) and from Bachem (Bubendorf, Switzerland), respectively. lZ5I-labeled IGF-I and IGF-I1 were prepared by the chloramine T method at a specific activity or 40-80 pCi/pg (Zapf et al., 1978). Leupeptin, chloroquine, phenanthroline, crude bovine serum albumin (BSA) (Cohn's fraction V), deoxyribonuclease I (DNAse I), and soybean trypsin inhibitor were purchased from Sigma. Antibody to glial fibrillary acidic protein (GFAP) was purchased from Dacopatts (Copenhagen, Denmark). Dulbecco's modified Eagle (DME) medium, fetal calf serum (FCS), and trypsin-EDTA were purchased from Gibco. Cell protein was measured by the Bio-Rad protein assay (Bradford, 1976).
15
bation in the presence 0.1 pM IGF-I or IGF-11, and amounted to about 10% of total tracer binding. In parallel, the number of cells in the wells was determined by counting in a standard hemocytometer chamber.
Internalization of IGF-I and IGF-I1 Internalized IGF was determined by the acid-wash technique (Haigler et al., 1980) with modifications (Nielsen et al., 1991). Astrocytes were incubated in KRH-buffer with albumin with ['251]IGF-I or [ '251]IGFI1 at a concentration of 0.2 nM for varying times at 37°C. Incubation was stopped by rapidly washing the cells twice with ice-cold PBS, followed by washing with acetic acid (0.2 M) and NaCl(0.5 M) at pH 3.5 for 5 min at 4°C. The acid-wash solution was collected, and the radioactivity representing surface-bound IGF-I or IGF-I1 was counted in a gamma counter. Finally, the cells were washed once with ice-cold PBS before being harvested with NaOH (0.2 M) and the radioactivity representing Cell Cultures internalized IGF-I or IGF-I1 was counted. The acid-senAstrocyte cultures were prepared from brains of sitive and acid-resistant fractions of cell-bound IGF were neonatal rats as described by Schousboe et al. (1975) corrected for nonspecific binding determined by incubawith modifications. The brains were taken out, freed of tion of cells in the presence of 0.1 pM IGF-I or IGF-11. meninges, and triturated with a 10-ml pipette in NaCl The nonspecifically bound tracers determined by incu(0.9 g/L), HEPES (25 mM) at pH 7.4 with trypsin (0.5 bation with 0.1 pM cold IGF-I or IGF-I1 amounted to g/L), and EDTA (0.2 g/L). When the tissue was disso- 10-15% of total surface-bound or internalized radioacciated, an equal volume of DME medium, with HEPES tivity. (25 mM), DNAse I (0.44 mg/ml), soybean trypsin inhibitor (0.55 mg/ml), and MgSO, (50 mM), at pH 7.4, Degradation of IGF-I and IGF-I1 was added. The cells that remained in solution after 5 Degradation of internalized IGF-I or IGF-I1 was min were pelleted and resuspended in culture medium determined in a pulse-chase experiment as described precomposed of DME medium with glucose (10 mg/ml), viously (Nielsen et al., 1991). Briefly, astrocytes were glutamine (0.1 mg/ml), penicillin (50 pU/ml), strepto- incubated in DME medium containing albumin (10 mM) mycin (50 pg/ml), supplemented with 20% FCS. The and HEPES (25 nM) at pH 7.4 with 0.6 nmol/L 1251cells were grown to confluency in 75-cm2 culture flasks labeled IGF-I or IGF-I1 for 60 min at 37°C. Extracellular after 1-2 weeks before they were trypsinized and trans- and surface-bound radioactivity was removed by washferred into 24-well multidishes for experiments. Immu- ing and acid extraction of the cells as described by Levy nocytochemical staining for GFAP showed that >97% of and Olefsky (1987). The cells were then reincubated in the cells stained positively. DME medium with HEPES and albumin with the addition of 0.6 nM unlabeled IGF-I or IGF-I1 for 3 hr at Receptor Binding of IGF-I and IGF-I1 37°C. The incubation medium was collected, and the Receptor-bound IGF-I and IGF-I1 on the cell sur- cells were extracted with HCl (0.1 M), Triton X-100 face was measured as described previously by Nielsen et (0.1% v/v), and albumin (1 mg/ml) for 90 min at 4°C. al. (1991). Briefly, monolayers of astrocytes at the in- The '251 activity released to the medium or extracted dicated densities in 24-well multidishes were incubated from the cells was analyzed by gel filtration on Sephadex in Krebs-Ringer-HEPES (KRH) buffer containing G-50 fine or by precipitation with trichloroacetic acid HEPES (25 mM, pH 7.4) and albumin (10 mg/ml) with (TCA) (0.1 g/ml). 0.15 nM [1251]IGF-Ior ['251]IGF-II. After 5 hr at 4"C, when steady state was reached, the incubation medium RESULTS was removed and the cells washed twice with ice-cold phosphate-buffered saline (PBS). Surface-bound radio- Internalization of IGF-I and IGF-I1 in Astrocytes The time courses of surface-bound and internalized activity was harvested with 0.2 mol/L NaOH and ['251]IGF-I and ['251]IGF-II in astrocytes at 37°C are counted in a gamma counter. The data were corrected for shown in Figure 1. IGF-I was rapidly bound to cellnonspecifically bound radioactivity, determined by incu-
16
Auletta et al. INTERNALIZATIONOF 1251-IGF-I AND1251-IGF-II BY FETAL RAT BRAIN ASTROCYTES AT 37'C.
-
6 1251-~~~-~
6
V.
IGFl
V,
A.
1
V.
IGFl
4
4
Vt
i
B
L
20
40
60
80
20
40
60
80
IGF- 11
8 0
60
120
180
TIME (min)
Fig. I . Internalization of IGF-I and IGF-I1 in astrocytes. Cells were incubated at 37°C for the indicated times with 0.2 nM of '2'1-labeled IGF-I (A) or IGF-I1 (B). The specific activities of ['2'I]IGF-I and ['2'1]IGF-II were 60 and 45 pCi/pg, respectively. Incubation was stopped by removal of the medium and washing with ice-cold PBS. Surface-bound "'I-activity ( 0 ) was released by incubation of the cells for 5 min at 4°C with 0.2 M acetic acid and 0.5 M NaCI, followed by extraction of internalized '251-activity ( 0 ) in 0.2 M NaOH (see Materials and Methods). Radioactivity was measured, corrected for nonspecific binding and expressed as cpm per p g cell protein. Data are mean of three experiments.
surface receptors and reached maximal binding after 30 min of incubation (Fig. 1A). This was accompanied by a slower continuous rise in internalized IGF-I that reached a maximum after about 30 min. At this time, approximately 40% of cell-associated ['251]IGF-I was acid resistant. Both surface-bound and internalized IGF-I declined gradually during the next 2 hr of incubation. Surface binding of ['251]IGF-II reached a maximal value after 15 min, declining to a constant level after 1 hr (Fig. 1B). After a brief lag time of about 5 min, the internalization of IGF-I1 increased rapidly to a maximum after 30 min, when about 80% of cell-associated [ '251]IGF-II was acid resistant. This was followed by a slight decline in internalized ICF-I1 over the following 2 h. During the 3-hr incubation, about 20% of ['251]IGF-I and ['251]IGFI1 in the medium was degraded as determined by the increase in TCA-soluble radioactivity (data not shown). The decrease in the concentrations of intact IGF-I and IGF-I1 in the medium may account for the decline in cell-associated radioactivity from 1 to 3 hr of incubation.
Volume (ml)
Fig. 2 . Degradation of internalized IGF-I and IGF-11. Astrocytes were incubated in culture medium without insulin containing albumin and HEPES at pH 7.4 for 60 min at 37°C with 0.6 nM ['251]IGF-I (A,B) or ['2'I]IGF-II (C,D). After washing 5 times with PBS at 4°C to remove extracellular IGF-I or IGF-11, the cells were acid-extracted to remove surface-bound peptide by incubation with KRH-buffer with albumin titrated at pH 3.5 for 10 min at 4°C. The cells were washed three more times with acidic KRH-buffer and three times with PBS at 4°C. During this extraction 99% of the surface-bound radioactivity was released (see Materials and Methods). The cells were reincubated with fresh culture medium without insulin containing HEPES and albumin at pH 7.4 for 3 hr at 37°C in the presence of 0.6 nM IGF-I or IGF-11. The incubation medium was collected and the cells were extracted with 0.1 M HCI, 0.1% Triton X-100, and 1 mg/ml albumin. The '251-activity released to the medium (A,C) or extracted from the cells (B,D) was analyzed by gel filtration on Sephadex G-50 fine and expressed as the percentage of cell-associated IGF-I or IGF-I1 determined as the sum of released and extracted radioactivity. The arrows indicate the void volume (VJ, IGF-I or IGF-I1 and the salt volume (V,). The data are from an experiment representative of two others with similar results.
Degradation of Internalized IGF-I and IGF-I1 The nature of ['251]IGF-I and [ '251]IGF-II, which have been internalized by astrocytes, was investigated in pulse-chase experiments and analysis of cell-associated radioactivity by gel filtration, as shown in Figure 2. It
Endocytosis of IGF-I and IGF-I1 in Astrocytes
17
TABLE I. Effect of Lysosomal and Protease Inhibitors on Surface-Binding,Internalization, and Degradation of IGF-I and IGF-I1 in Cultured Astrocytest'* Inhibitor
Concn (mM)
Surface-bound
[L251]IGF-Icpm/p,g protein (% of control) Control 3.9 t 0.4 (100) Chloroquine 0.1 4.6 t 0.5 (117) 1.o 8.3 t 1.9" (212) Phenanthroline Leupeptin 0.2 3.5 IT 0.2 (89) [L251]IGF-IIcpm/p,g protein (% of control) Control 2.4 t 0.2 (100) Chloroquine 0.1 3.7 f 0.4b (154) Phenanthroline 1.o 4.5 f 0.4' (187) Leupeptin 0.2 2.6 t 0.2 (108)
Internalized 3.0 6.1 3.9 3.6
t 0.4 IT 0.6" t 1.2 t 0.4
(100) (203) (130) (120)
10.8 t 1.0 (100) 13.5 1.6 (125) 25.4 3.7" (235) 15.9 2 1.6b (147)
* *
Degraded 1.6 f 0.2 1.1 IT 0.1" 1.2 f 0.1 1.4 f 0.1
(100) (68) (75) (88)
7.6 f 0.7 (100) 4.4 k 1.4" (58) 5.6 0.5" (74) 7.1 f 0.9 (93)
*
'Astrocytes were preincubated with lysosomal or protease inhibitors for 30 rnin at 37°C. ['251]IGF-I or [1251]IGF-IIat a concentration of 0.6 nM was added and the incubation continued for 60 min (determination of degradation) or 180 min (determination of surface binding and internalization) according to the protocols described under Materials and Methods. Degraded IGF-I and IGF-I1 was determined as trichloroacetic acid-soluble Iz5I activity. Data are mean of three experiments with SD. *Student's t-test was used to estimate the significance of the difference between mean values of control and inhibitor-treated cells. "P
Receptor-Mediated Endocytosis and Degradation of Insulin-Like Growth Factor I and 11 in Neonatal Rat Astrocytes M. Auletta, F.C. Nielsen, and S. Gammeltoft Department of Clinical Chemistry, Bispebjerg Hospital, Copenhagen, Denmark Lund et al., 1986; Saraet al., 1986; Rotwein et al., 1988; Stylianopoulou et al., 1988). Two types of IGF receptors are distributed throughout the fetal and adult mammalian CNS (Sara et al., 1983; Gammeltoft et al., 1985). Recent studies have demonstrated that astrocytes have receptors for IGF-I and IGF-I1 and that IGFs stimulate DNA synthesis via activation of the IGF-I receptor tyrosine kinase (Ballotti et al., 1987; Han et al., 1987; Shemer et al., 1987). On the basis of the finding that neonatal rat astrocytes express both IGF-I and IGF-I1 (Ballotti et al., 1987; Rotwein et al., 1988), it has been suggested that IGFs stimulate the clonal expansion of astrocytes in neonatal rat brain by an autocrine or paracrine mechanism (Gammeltoft et al., 1988). This raises the question of whether IGF-I and IGF-11, which are produced by astrocytes and interact with cell-surface receptors, are also removed and processed by these cells. Receptor-mediated endocytosis of polypeptide hormones, growth factors, and neuropeptides represents the major pathway in the breakdown of bioactive substances (Gammeltoft, 1991). In various cell types, IGF-I and IGF-I1 are internalized and degraded by interaction with IGF-I receptors and mannose 6-phosphate/IGF-I1 (Man6-P/IGF-11) receptors, respectively (Bar et al., 1986; Banskota et al., 1986; Frank et al., 1986; Hachiya et al., 1986; Oka et al., 1985; Schalch et al., 1985; Nielsen et al., 1991. In the present study, we show that IGF-I and IGF-I1 are processed by two distinct pathways of receptor-meKey words: insulin-like growth factor, IGF-I recep- diated endocytosis and degradation in neonatal rat astrotor, IGF-I1 receptor, Man-6-P receptor, endocytosis, cytes. Furthermore, the expression of IGF-I receptors IGF degradation, central nervous system, glial cells and Man-6-P/IGF-I1 receptors on the cell surface seems to be regulated by independent mechanisms. INTRODUCTION
Receptor-mediated internalization and degradation of insulin-like growth factors, IGF-I and IGF-11, were studied in primary cultures of neonatal rat astrocytes. Surface-bound IGF-I1 was rapidly internalize& and 80% of cell-associated radioactivity was located intracellularly after 30 min. IGF-I was internalized at a slower rate, and only 40% of cell-associated radioactivity was inside the cell after 30 min. A pulse-chase experiment demonstrated that 55% and 70% of internalized IGF-I and IGF-11, respectively, was degraded to free amino acids after a 3-hr chase. Lysosomal and protease inhibitors had different effects on the binding, internalization, and processing of IGF-I and IGF-11. Inhibition of lysosomal acidification by chloroquine increased the amounts of surface-bound IGF-I1 and intracellular IGF-I and reduced the degradation of IGF-I. The chelating agent phenanthroline increased the surface binding of IGFI and IGF-I1 and internalization of IGF-I1 and reduced the degradation of IGF-I and IGF-11. Finally, receptor-bound IGF-I1 on the cell surface was decreased with increasing cell density, whereas IGF-I binding was unaltered. Our data suggest that cellsurface expression of IGF-I receptors and IGF-I1 receptors is regulated by different mechanisms and that receptor-bound IGF-I and IGF-I1 are trafficked and processed by different intracellular pathways in neonatal rat astrocytes.
Received January 18, 1991; accepted May 29, 1991.
Insulin-like growth factors (IGF) I and 11 are polypeptides structurally similar to insulin that act as mitogens in various fetal and adult tissues (Froesch et al., 1985; Gammeltoft, 1989). In the central nervous system (CNS) of fetal and adult humans and rat, gene and protein expression of IGF-I and IGF-I1 has been demonstrated (Haselbacher et al., 1985; Brown et al., 1986; 0 1992 Wiley-Liss, Inc.
Address reprint requests to Dr. Steen Gammeltoft, Department of Clinical Chemistry, Bispebjerg Hospital, DK-2400 Copenhagen NV, Denmark. M. Auletta is presently at Fourth Division of Internal Medicine, Second Faculty of Medicine and Surgery, University of Napoli, Italy. F.C. Nielsen is presently at the Department of Clinical Chemistry, Rigshospitalet, University of Copenhagen, Denmark.
Endocytosis of IGF-I and IGF-I1 in Astrocytes MATERIALS AND METHODS Materials Recombinant human IGF-I and IGF-I1 were purchased from Boehringer (Mannheim, West Germany) and from Bachem (Bubendorf, Switzerland), respectively. lZ5I-labeled IGF-I and IGF-I1 were prepared by the chloramine T method at a specific activity or 40-80 pCi/pg (Zapf et al., 1978). Leupeptin, chloroquine, phenanthroline, crude bovine serum albumin (BSA) (Cohn's fraction V), deoxyribonuclease I (DNAse I), and soybean trypsin inhibitor were purchased from Sigma. Antibody to glial fibrillary acidic protein (GFAP) was purchased from Dacopatts (Copenhagen, Denmark). Dulbecco's modified Eagle (DME) medium, fetal calf serum (FCS), and trypsin-EDTA were purchased from Gibco. Cell protein was measured by the Bio-Rad protein assay (Bradford, 1976).
15
bation in the presence 0.1 pM IGF-I or IGF-11, and amounted to about 10% of total tracer binding. In parallel, the number of cells in the wells was determined by counting in a standard hemocytometer chamber.
Internalization of IGF-I and IGF-I1 Internalized IGF was determined by the acid-wash technique (Haigler et al., 1980) with modifications (Nielsen et al., 1991). Astrocytes were incubated in KRH-buffer with albumin with ['251]IGF-I or [ '251]IGFI1 at a concentration of 0.2 nM for varying times at 37°C. Incubation was stopped by rapidly washing the cells twice with ice-cold PBS, followed by washing with acetic acid (0.2 M) and NaCl(0.5 M) at pH 3.5 for 5 min at 4°C. The acid-wash solution was collected, and the radioactivity representing surface-bound IGF-I or IGF-I1 was counted in a gamma counter. Finally, the cells were washed once with ice-cold PBS before being harvested with NaOH (0.2 M) and the radioactivity representing Cell Cultures internalized IGF-I or IGF-I1 was counted. The acid-senAstrocyte cultures were prepared from brains of sitive and acid-resistant fractions of cell-bound IGF were neonatal rats as described by Schousboe et al. (1975) corrected for nonspecific binding determined by incubawith modifications. The brains were taken out, freed of tion of cells in the presence of 0.1 pM IGF-I or IGF-11. meninges, and triturated with a 10-ml pipette in NaCl The nonspecifically bound tracers determined by incu(0.9 g/L), HEPES (25 mM) at pH 7.4 with trypsin (0.5 bation with 0.1 pM cold IGF-I or IGF-I1 amounted to g/L), and EDTA (0.2 g/L). When the tissue was disso- 10-15% of total surface-bound or internalized radioacciated, an equal volume of DME medium, with HEPES tivity. (25 mM), DNAse I (0.44 mg/ml), soybean trypsin inhibitor (0.55 mg/ml), and MgSO, (50 mM), at pH 7.4, Degradation of IGF-I and IGF-I1 was added. The cells that remained in solution after 5 Degradation of internalized IGF-I or IGF-I1 was min were pelleted and resuspended in culture medium determined in a pulse-chase experiment as described precomposed of DME medium with glucose (10 mg/ml), viously (Nielsen et al., 1991). Briefly, astrocytes were glutamine (0.1 mg/ml), penicillin (50 pU/ml), strepto- incubated in DME medium containing albumin (10 mM) mycin (50 pg/ml), supplemented with 20% FCS. The and HEPES (25 nM) at pH 7.4 with 0.6 nmol/L 1251cells were grown to confluency in 75-cm2 culture flasks labeled IGF-I or IGF-I1 for 60 min at 37°C. Extracellular after 1-2 weeks before they were trypsinized and trans- and surface-bound radioactivity was removed by washferred into 24-well multidishes for experiments. Immu- ing and acid extraction of the cells as described by Levy nocytochemical staining for GFAP showed that >97% of and Olefsky (1987). The cells were then reincubated in the cells stained positively. DME medium with HEPES and albumin with the addition of 0.6 nM unlabeled IGF-I or IGF-I1 for 3 hr at Receptor Binding of IGF-I and IGF-I1 37°C. The incubation medium was collected, and the Receptor-bound IGF-I and IGF-I1 on the cell sur- cells were extracted with HCl (0.1 M), Triton X-100 face was measured as described previously by Nielsen et (0.1% v/v), and albumin (1 mg/ml) for 90 min at 4°C. al. (1991). Briefly, monolayers of astrocytes at the in- The '251 activity released to the medium or extracted dicated densities in 24-well multidishes were incubated from the cells was analyzed by gel filtration on Sephadex in Krebs-Ringer-HEPES (KRH) buffer containing G-50 fine or by precipitation with trichloroacetic acid HEPES (25 mM, pH 7.4) and albumin (10 mg/ml) with (TCA) (0.1 g/ml). 0.15 nM [1251]IGF-Ior ['251]IGF-II. After 5 hr at 4"C, when steady state was reached, the incubation medium RESULTS was removed and the cells washed twice with ice-cold phosphate-buffered saline (PBS). Surface-bound radio- Internalization of IGF-I and IGF-I1 in Astrocytes The time courses of surface-bound and internalized activity was harvested with 0.2 mol/L NaOH and ['251]IGF-I and ['251]IGF-II in astrocytes at 37°C are counted in a gamma counter. The data were corrected for shown in Figure 1. IGF-I was rapidly bound to cellnonspecifically bound radioactivity, determined by incu-
16
Auletta et al. INTERNALIZATIONOF 1251-IGF-I AND1251-IGF-II BY FETAL RAT BRAIN ASTROCYTES AT 37'C.
-
6 1251-~~~-~
6
V.
IGFl
V,
A.
1
V.
IGFl
4
4
Vt
i
B
L
20
40
60
80
20
40
60
80
IGF- 11
8 0
60
120
180
TIME (min)
Fig. I . Internalization of IGF-I and IGF-I1 in astrocytes. Cells were incubated at 37°C for the indicated times with 0.2 nM of '2'1-labeled IGF-I (A) or IGF-I1 (B). The specific activities of ['2'I]IGF-I and ['2'1]IGF-II were 60 and 45 pCi/pg, respectively. Incubation was stopped by removal of the medium and washing with ice-cold PBS. Surface-bound "'I-activity ( 0 ) was released by incubation of the cells for 5 min at 4°C with 0.2 M acetic acid and 0.5 M NaCI, followed by extraction of internalized '251-activity ( 0 ) in 0.2 M NaOH (see Materials and Methods). Radioactivity was measured, corrected for nonspecific binding and expressed as cpm per p g cell protein. Data are mean of three experiments.
surface receptors and reached maximal binding after 30 min of incubation (Fig. 1A). This was accompanied by a slower continuous rise in internalized IGF-I that reached a maximum after about 30 min. At this time, approximately 40% of cell-associated ['251]IGF-I was acid resistant. Both surface-bound and internalized IGF-I declined gradually during the next 2 hr of incubation. Surface binding of ['251]IGF-II reached a maximal value after 15 min, declining to a constant level after 1 hr (Fig. 1B). After a brief lag time of about 5 min, the internalization of IGF-I1 increased rapidly to a maximum after 30 min, when about 80% of cell-associated [ '251]IGF-II was acid resistant. This was followed by a slight decline in internalized ICF-I1 over the following 2 h. During the 3-hr incubation, about 20% of ['251]IGF-I and ['251]IGFI1 in the medium was degraded as determined by the increase in TCA-soluble radioactivity (data not shown). The decrease in the concentrations of intact IGF-I and IGF-I1 in the medium may account for the decline in cell-associated radioactivity from 1 to 3 hr of incubation.
Volume (ml)
Fig. 2 . Degradation of internalized IGF-I and IGF-11. Astrocytes were incubated in culture medium without insulin containing albumin and HEPES at pH 7.4 for 60 min at 37°C with 0.6 nM ['251]IGF-I (A,B) or ['2'I]IGF-II (C,D). After washing 5 times with PBS at 4°C to remove extracellular IGF-I or IGF-11, the cells were acid-extracted to remove surface-bound peptide by incubation with KRH-buffer with albumin titrated at pH 3.5 for 10 min at 4°C. The cells were washed three more times with acidic KRH-buffer and three times with PBS at 4°C. During this extraction 99% of the surface-bound radioactivity was released (see Materials and Methods). The cells were reincubated with fresh culture medium without insulin containing HEPES and albumin at pH 7.4 for 3 hr at 37°C in the presence of 0.6 nM IGF-I or IGF-11. The incubation medium was collected and the cells were extracted with 0.1 M HCI, 0.1% Triton X-100, and 1 mg/ml albumin. The '251-activity released to the medium (A,C) or extracted from the cells (B,D) was analyzed by gel filtration on Sephadex G-50 fine and expressed as the percentage of cell-associated IGF-I or IGF-I1 determined as the sum of released and extracted radioactivity. The arrows indicate the void volume (VJ, IGF-I or IGF-I1 and the salt volume (V,). The data are from an experiment representative of two others with similar results.
Degradation of Internalized IGF-I and IGF-I1 The nature of ['251]IGF-I and [ '251]IGF-II, which have been internalized by astrocytes, was investigated in pulse-chase experiments and analysis of cell-associated radioactivity by gel filtration, as shown in Figure 2. It
Endocytosis of IGF-I and IGF-I1 in Astrocytes
17
TABLE I. Effect of Lysosomal and Protease Inhibitors on Surface-Binding,Internalization, and Degradation of IGF-I and IGF-I1 in Cultured Astrocytest'* Inhibitor
Concn (mM)
Surface-bound
[L251]IGF-Icpm/p,g protein (% of control) Control 3.9 t 0.4 (100) Chloroquine 0.1 4.6 t 0.5 (117) 1.o 8.3 t 1.9" (212) Phenanthroline Leupeptin 0.2 3.5 IT 0.2 (89) [L251]IGF-IIcpm/p,g protein (% of control) Control 2.4 t 0.2 (100) Chloroquine 0.1 3.7 f 0.4b (154) Phenanthroline 1.o 4.5 f 0.4' (187) Leupeptin 0.2 2.6 t 0.2 (108)
Internalized 3.0 6.1 3.9 3.6
t 0.4 IT 0.6" t 1.2 t 0.4
(100) (203) (130) (120)
10.8 t 1.0 (100) 13.5 1.6 (125) 25.4 3.7" (235) 15.9 2 1.6b (147)
* *
Degraded 1.6 f 0.2 1.1 IT 0.1" 1.2 f 0.1 1.4 f 0.1
(100) (68) (75) (88)
7.6 f 0.7 (100) 4.4 k 1.4" (58) 5.6 0.5" (74) 7.1 f 0.9 (93)
*
'Astrocytes were preincubated with lysosomal or protease inhibitors for 30 rnin at 37°C. ['251]IGF-I or [1251]IGF-IIat a concentration of 0.6 nM was added and the incubation continued for 60 min (determination of degradation) or 180 min (determination of surface binding and internalization) according to the protocols described under Materials and Methods. Degraded IGF-I and IGF-I1 was determined as trichloroacetic acid-soluble Iz5I activity. Data are mean of three experiments with SD. *Student's t-test was used to estimate the significance of the difference between mean values of control and inhibitor-treated cells. "P
