0013-7227/90/1266-2998$02.00/0 Endocrinology Copyright © 1990 by The Endocrine Society

Vol. 126, No. 6 Printed in U.S.A.

Characterization of Type I and Type II Insulin-Like Growth Factor Receptors in an Intestinal Epithelial Cell Line* JUNG H. Y. PARK, JON A. VANDERHOOF, DARCY BLACKWOOD, AND RICHARD G. MACDONALD Department of Pediatrics, Creighton University (J.H.Y.P., J.A.V.), Departments of Pediatrics (J.H.Y.P., J.A.V., D.B.) and Biochemistry (R.G.M.) University of Nebraska Medical Center, Omaha, Nebraska 68198

ABSTRACT. Insulin-like growth factors (IGFs) and insulin stimulate DNA and protein synthesis in IEC-6 cells (an intestinal epithelial cell line) grown in a chemically defined medium. 1GF-I stimulates proliferation of IEC-6 cells at a lower concentration (ED60 = 1.6 nM) than either insulin or IGF-II. To gain insight into the mechanisms by which IGFs stimulate IEC-6 cell growth, we have examined the characteristics of specific IGF receptors on IEC-6 cells. Binding of 125I-IGF-I and 125I-IGF-II to IEC-6 monolayers was analyzed by incubation with various concentrations (0.2 nM to 0.5 MM) of radiolabeled IGFs for 16 h at 3 C. Scatchard plots of 125I-IGF-I binding were linear, suggesting a single class of binding sites with KD = 3.1 ± 0.35 nM and Bmax = 50.7 ± 6 fmol/106 cells. IGF-II was potent in displacing 125I-IGF-I (Ki= 8.1 ± 0.85 nM), but insulin had little effect. Affinity cross-linking with 126I-IGF-I followed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed three bands with Mr of 270,000, 245,000, and 133,000, and the major band was the 133,000 species. Labeling of the 133,000 and 270,000

T

HE mucosa of the small intestine represents a distinctive population of cells that perpetually regenerate themselves in an effort to balance cell loss through exfoliation by the process of cellular proliferation and growth (1, 2). The mechanisms which control cell regeneration are unknown. This is due, in part, to the multiple factors involved in the regulation of intestinal epithelial cell proliferation and differentiation. These factors include the general nutritional status of animals, luminal nutrients, endocrine factors, paracrine factors, and celhcell interactions (1, 2). Therefore, in order to study the detailed mechanisms by which intestinal mucosa is regulated, it is necessary to establish an experimental model system in which the effects of specific substances can be assessed independently of confounding variables.

Received December 7,1989. Address all correspondence and reprint requests to: Dr. Jung H. Y. Park, Swanson Center Room 3049, 600 South 42nd Street, Omaha, Nebraska, 68198-5160. * This work was supported by a grant from Caremark, Inc., Deerfield, IL, and by the University of Nebraska Seed Grant Program.

bands was >80% inhibited by 10 7 M unlabeled IGF-I, less potently inhibited by IGF-II and not at all by insulin. These results suggest that the 133,000 band represents the a-subunit of the type I IGF receptor. Scatchard plots of 126I-IGF-II binding to IEC-6 cell monolayers were curvilinear, suggesting two classes of binding sites: high affinity, low capacity sites, KD = 0.87 ± 0.08 nM and Bmax = 28 ± 2.5 fmol/106 cells; low affinity, high capacity sites, KD = 60 = ± 8.8 nM and Bmax = 1780 ± 230 fmol/ 106 cells. Neither IGF-I nor insulin was effective in inhibiting 125 I-IGF-II binding. Affinity cross-linking with 125I-IGF-II labeled predominantly a 245,000 band, suggesting that this species is the type II receptor. A band with Mr 131,000 was barely detectable with 125I-insulin. These results indicate that IEC-6 cells have abundant quantities of the type I and II IGF receptors and few insulin receptors, suggesting that the mitogenic effect of IGFs is mediated through the type I IGF receptor. (Endocrinology 126: 2998-3005, 1990)

The best model would be cultures of cells derived from the small intestine under chemically defined experimental conditions and uncontaminated by different cell types. Proliferation of intestinal epithelium has been examined in IEC-6 cells, an intestinal cell line derived from rat jejunal crypts (3, 4). We have previously developed a chemically defined serum-free medium that supports the metabolic and survival needs of IEC-6 cells.1 Utilizing this serum-free medium we have observed that insulin and the insulin-like growth factors (IGFs), IGFI and IGF-II, stimulate IEC-6 cell growth. Among these three growth factors, IGF-I was the most effective. IGFs are mitogenic polypeptides with structural similarity to proinsulin (5, 6). The initial event in the action of these three polypeptides on target cells is believed to be their binding to specific receptors at the cell surface (6-8). The type I IGF receptor is similar to the insulin receptor in its structure. It is a heterotetramer of a2^2 1 Park JHY, Harty RF, Joekel CS, Blackwood D, Vanderhoof JA IGF-I stimulates intestinal epithelial cell growth in serum-free culture. Submitted for publication.

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IGF RECEPTORS IN AN INTESTINAL EPITHELIAL CELL LINE form and contains tyrosine-specific protein kinase activity within its intracellular domain (6, 7, 9, 10). Both the IGF-I receptor and the insulin receptor have been firmly established to mediate the biological effects of their cognate ligands in target cells through activation of the intrinsic tyrosine kinase (7, 8, 11). By contrast, the type II IGF receptor is composed of a single polypeptide with a Mr of 270,000 (7, 9, 10, 12). Recent experiments have demonstrated that the IGF-II receptor is the same as the cation-independent mannose-6-phosphate receptor (1216). As such, the IGF-II receptor is capable of binding both IGF-II and lysosomal enzymes at apparently distinct binding sites within the receptor's extracellular domain (14-18). The functional consequences of IGF-II binding to this receptor are currently a matter of controversy, particularly since the receptor does not contain tyrosine kinase activity (7, 12). Previous studies have demonstrated considerable heterogeneity in the distribution of receptors for insulin and the IGFs among mammalian tissues (6, 10). Furthermore, it is often difficult to determine which receptor type may mediate a particular biological response to the IGFs because of the overlapping ligand binding specificities of the receptors (6, 19). The precise biological roles of IGFs and insulin and the characteristics of their receptors in the intestinal mucosa have not been established. In an effort to gain insight into the mechanism by which these growth factors regulate proliferation of IEC-6 cells, the present studies have investigated the properties of specific IGF and insulin receptors on IEC-6 cells.

Materials and Methods Materials Dulbecco's modified Eagle Medium (DMEM), F-12 nutrient mixture (F12), dialyzed fetal bovine serum (FBS), trypsinEDTA, and penicillin-streptomycin were obtained from GIBCO (Grand Island, NY). Tissue culture dishes and 6-well plates were purchased from Becton Dickinson (Lincoln Park, NJ). RIA grade BSA was purchased from Sigma Chemical (St. Louis, MO). Recombinant human IGF-I and IGF-II, and porcine insulin for competitive binding studies were kindly provided by Mrs. M. H. Niedenthal (Lilly Research Laboratories, Indianapolis, IN). IEC-6 cells, originally established in culture by Quaroni et al. (3), were purchased from the American Type Culture Collection (ATCC CRL 1592, Rockville, MD). Porcine m I-insulin, monoiodinated receptor grade (specific activity, 2200 Ci/mmol), was purchased from New England Nuclear (Boston, MA). 125I-[Thr59]IGF-I (specific activity, 2000 Ci/ mmol) was obtained from Amersham (Arlington Heights, IL). IGF-II was radioiodinated by using an immobilized lactoperoxidase/glucose oxidase reagent (Enzymobeads, Bio-Rad, Richmond, CA) to a specific activity of 45-70 Ci/g. Dissuccinimidyl suberate (DSS) was purchased from Pierce (Rockford, IL).

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Unless otherwise stated, all other chemicals were obtained from Sigma. Cell culture For routine maintenance, monolayers of IEC-6 cells were grown in DMEM/F12 (3:1) containing 5% FBS, 10 Mg/ml insulin, 20 U/ml penicillin, and 20 ii%/m\ streptomycin in tissue culture dishes (100 mm). Cells between the 16th and 19th passage were used for these experiments. Cells were maintained and subcultured as described by Quaroni and May (4). IGF and insulin binding studies in IEC-6 cell monolayers IEC-6 cells were grown in 6-well plates to 80% confluency in DMEM/F12 plus 5% FBS and 10 /ig/ml insulin. The monolayers were washed twice with Hanks' balanced salt solution and serum starved for 24 h in DMEM/F12 containing 50 ng/ ml dexamethasone, 5 Mg/ml transferrin, and 0.5 mg/ml BSA. After the 24 h serum starvation, IEC-6 cells were washed three times with DMEM/F12 containing 25 mM HEPES and 1% BSA (binding buffer). The monolayers were incubated for 16 h at 3 C with 0.2 nM 125I-IGF-I, 125I-IGF-II or 125I-insulin and various concentrations (0-5 x 10~7 M) of unlabeled IGF-I, IGFII or insulin in binding buffer. At the end of the overnight incubation, the binding buffer was aspirated and the plates were washed three times with binding buffer at 3 C. The surface-bound 125I-IGF-I or 125I-IGF-II were counted in a gamma counter following solubilization of the cells with 0.4 N NaOH. Nonspecific binding, defined as the radioactivity bound in the presence of 0.5 nM unlabeled peptide, was subtracted from total binding to obtain specific binding. Nonspecific binding represented 10-20% of the maximal binding. In each experiment, replicate wells were used to determine cell number using a hemacytometer. The data were initially analyzed by the method of Scatchard (20) using the computer program, EBDA, as described by McPherson (21). Because the Scatchard plots of saturation data for 125I-IGF-II were curvilinear, the data were fitted to a two-site model by the method of Munson and Rodbard (22) using the computer program, LIGAND. Membrane isolation For receptor-labeling experiments, IEC-6 cells were grown in 100 mm dishes and serum starved as described above. The cells were then chilled to 3 C, scraped, and membranes were obtained by homogenization of cells in 0.25 M sucrose, 10 mM Tris-HCl, 1 mM EDTA, 1 mM phenylmethylsulfonyl fluoride (PMSF), pH 7.4, with a tight fitting Dounce homogenizer (3 X 20 strokes). The homogenate was centrifuged at 2,500 x g for 12 min, and the resulting supernatant was centrifuged at 25,000 x g for 45 min. The pellets were resuspended in 10 mM TrisHCl, 1 mM EDTA, 1 mM PMSF, pH 7.4, and centrifuged at 25,000 x g for 40 min. The final pellets were resuspended in a small volume of the same buffer and stored at -20 C until needed. Protein was assayed by the Bio-Rad method using human 7-globulin as standard.

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IGF RECEPTORS IN AN INTESTINAL EPITHELIAL CELL LINE

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Affinity labeling of receptors on isolated membranes Affinity labeling of the IGF and insulin receptors in the membranes using DSS was carried out by a modification of previously described procedures (10). Membranes were suspended in Krebs-Ringer phosphate buffer, pH 7.4, containing 1% BSA and antiproteases to a concentration of 1 mg membrane protein per ml (0.2 ml total volume). The antiproteases used were 10 ng/ral leupeptin, 10 ixg/ml antipain, 80 fig/ml benzamidine, 20 Mg/ml aprotinin, 10 fig/ml pepstatin, and 0.5 mM PMSF. The suspensions were incubated at 3 C for 18 h with 1-2 nM 125I-labeled hormones in the presence or absence of unlabeled hormones at the indicated final concentrations. DSS freshly dissolved in dimethyl sulfoxide was added to give a final concentration of 0.25 mM followed by a 15-min incubation on ice. The cross-linking reaction was quenched by addition of 0.5 ml of 0.1 M Tris-HCl, pH 7.4. The cross-linked membranes were isolated by centrifugation, dissolved in sample buffer, and electrophoresed on polyacrylamide slab gels according to Laemmli (23) after heating to 100 C for 6 min in the presence of 50 mM dithiothreitol. The gel was stained, destained, dried, and subjected to autoradiography using Dupont Cronex Lightning Plus intensifying screens. The molecular weight standards for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) were myosin (Mr = 200,000), 0galactosidase (116,250), phosphorylase b (92,400), BSA (66,200), and ovalbumin (45,000). The amount of radioactivity associated with the labeled receptor bands was measured by counting the corresponding gel fragments in a gamma counter.

CD

0

40

50

Bound, fmol/106 cells FIG. 1. Scatchard analysis of 125I-IGF-I binding to IEC-6 cell monolayers. Cell monolayers were incubated with various concentrations (2 x 1(T10 to 5 x 10"7 M) of 125I-IGF-I and then analyzed for specific 128IIGF-I binding. The inset depicts these same data plotted as a saturation binding curve. Each point represents an individual measurement made in a single experiment which is representative of four replicates.

120 T O

100

Results IGF and insulin binding studies in IEC-6 cell monolayers 125

Analysis of I-IGF-I binding isotherms to IEC-6 cell monolayers according to the method of Scatchard revealed linear plots consistent with a single class of noninteracting binding sites with KD of 3.1 ± 0.35 nM and Bmax of 51 ± 6 fmol/106 cells, which represents approximately 30,000 surface binding sites per cell (Fig. 1). The specificity of binding of 125I-IGF-I by IEC-6 cell monolayers is shown in Fig. 2. Binding of 125I-IGF-I was inhibited by unlabeled IGF-I with an apparent Ki of 2.7 ± 0.27 nM and by IGF-II with approximately 3-fold lower potency (Ki = 8.1 ± 0.85 nM). Insulin had little effect on 125 I-IGF-I binding to IEC-6 cell monolayers up to a concentration of 1 X 10"7 M. Insulin at 5 X 10~7 M inhibited 125I-IGF-I binding by 18%. These data are consistent with the hypothesis that 125I-IGF-I is binding primarily to a type I IGF receptor in IEC-6 cell monolayers. Scatchard plot analysis of 125I-IGF-II binding isotherms to IEC-6 cell monolayers was curvilinear, suggesting the presence of a heterogeneous receptor population (Fig. 3). Using a two-site model, the high binding affinity (KD = 0.87 ± 0.08 nM), low capacity sites (Bmax = 28 ± 2.5 fmol/106 cells or about 17,000 surface binding sites per cell) and the low affinity (KD = 60.2 ± 8.8 nM),

14

m 80 u 1—4 |X4 •-*

60

DU CO

40

o w

1*4

o 8

20 0

o IGF-I • IGF-II MNSULIN -9

-7

LOG PEPTIDE [M] FIG. 2. Competition by unlabeled IGF-I, IGF-II, and insulin for 125IIGF-I binding to IEC-6 cell monolayers. Binding assays were performed by incubation of cell monolayers with 0.2 nM 125I-IGF-I in the presence of increasing concentrations of unlabeled peptides as indicated. Specific binding of labeled IGF-I is expressed as a percentage of binding in the absence of any unlabeled peptide. Each point represents the mean of three to four duplicate experiments. Interassay variations were approximately 10-20%.

high capacity sites (Bmax = 1,780 ± 230 fmol/106 cells) could be identified. As shown in Fig. 4, binding of 125IIGF-II to IEC-6 cell monolayers was inhibited by unlabeled IGF-II with an apparent Ki of 21.2 ± 0.5 nM. Neither IGF-I nor insulin were effective in inhibiting radiolabeled IGF-II binding to IEC-6 cell monolayers. The characteristics of 125I-IGF-II binding to the high

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IGF RECEPTORS IN AN INTESTINAL EPITHELIAL CELL LINE

f1

80% inhibited by 100 nM IGF-I. IGF-II was less potent than IGF-I in its ability to inhibit labeling of either the 133,000 or 270,000 species; percent inhibition of the 133,000 band was 10% and 50% for 20 nM and 100 nM IGF-II, respectively. By contrast, unlabeled IGF-II inhibited labeling of the 245,000 species more efficiently than IGF-I; 20 nM IGFII completely blocked labeling of this species by 125I-IGFI (Fig. 5, lane D). Unlabeled insulin at 100 nM was totally

'•

OS.

125

MGF-I

Mr x 10"

40-

o IGF-II

° \

• IGF-I 20- • AINSUUN 0-

200-

1

1

1

1

-9

-8

-7

-6

LOG PEPTIDE [M] FIG. 4. Competition by unlabeled IGF-II, IGF-I, and insulin for 125IIGF-II binding to IEC-6 cell monolayers. Cells were incubated with 0.2 nM m I-IGF-II and increasing concentrations of unlabeled peptides as indicated. Specific binding of labeled IGF-II is expressed as a percentage of binding in the absence of any unlabeled peptide. Each point represents the means of three to four duplicate experiments. Interassay variations were approximately 10-20%.

affinity, low capacity sites are typical of the type II IGF receptor. The low affinity, high capacity binding component might represent IGF binding proteins bound to the cell surface or directly to the tissue culture plastic. In several experiments, we observed displaceable binding of 125I-IGF-II to cell-free culture wells that had been

67Unbbclcd pcplidc nM

-

20

100

20

100

100

FIG. 5. Affinity cross-linking of 125I-IGF-I to IEC-6 cell membranes. Membranes were incubated with 1 nM 125I-IGF-I in the presence of no unlabeled peptide (lane A) or the indicated concentrations of unlabeled IGF-I, IGF-II or insulin. The receptor-ligand complexes were crosslinked with DSS and electrophoresed on a 5% polyacrylamide gel after treatment with 50 mM dithiothreitol. An autoradiogram of the dried gel is shown. These data are representative of three replicate experiments.

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IGF RECEPTORS IN AN INTESTINAL EPITHELIAL CELL LINE 125

A

Endo • 1990 Vol 126 • No 6 l25

I-IGF-I B

C

D

E

I-Insulin

F

G

H

Mr x10' 3

Unlabeled peptide nM

-

IGF-II 20

100

IGF-II

IGF-I 20

100

20

100

-

100 M-6-P

125

FIG. 6. Affinity cross-linking of I-IGF-II to IEC-6 cell membranes. Membranes were incubated with 1 nM 125I-IGF-II in the presence of no unlabeled peptide (lane A) or the indicated concentrations of unlabeled peptides. Samples in lanes H and I were incubated with 5 mM mannose6-phosphate (M-6-P) in the absence (H) or presence (I) of 100 nM unlabeled IGF-II. The receptor-ligand complexes were cross-linked with DSS and electrophoresed on a 6% polyacrylamide gel after treatment with 50 mM dithiothreitol. An autoradiogram of the dried gel is shown. These data are representative of three replicate experiments.

ineffective in inhibiting the 125I-IGF-I labeling of any of these three bands (Fig. 5, lane F). Affinity cross-linking of 125I-IGF-II to isolated IEC-6 membranes resulted in nearly exclusive labeling of an Mr = 245,000 band (Fig. 6). Unlabeled IGF-II efficiently inhibited labeling of this species, whereas IGF-I and insulin at identical concentrations were without effect. Inclusion of 5 mM mannose-6-phosphate enhanced labeling of the Mr = 245,000 band (Fig. 6, lane H). Figure 7 shows results of affinity cross-linking with 2 nM 125I-IGF-I (A-D) or 2 nM 125I-insulin (E-H). With 2 nM 125I-IGF-I, the results were basically the same as those shown in Figure 5. With the identical concentration of 125I-insulin, a band with an apparent Mr of 131,000 was barely detectable (Fig. 7, lane E). Labeling of this species was partially inhibited by 100 nM concentrations of unlabeled insulin, IGF-I or IGF-II. Discussion The results of the present experiments clearly indicate that IEC-6 cells are equipped with both type I and type II IGF receptors. The following findings favor the existence of a distinct type I IGF receptor in IEC-6 cells: 1) 125 I-IGF-I binding to intact IEC-6 cell monolayers was specific; 2) the relative rank order of potency in displacing 125I-IGF-I binding was IGF-I > IGF-II»insulin; 3) affinity cross-linking with 125I-IGF-I followed by SDSPAGE under reducing conditions revealed the presence of a labeled species of apparent Mr = 133,000. The size

FIG. 7. Affinity cross-linking of

125

I-IGF-I or

l25

I-insulin to IEC-6 cell

monolayers. Lanes A-D: Membranes were incubated with 2 nM 12BIIGF-I in the presence of 100 nM unlabeled IGF-II (A), 100 nM IGF-I (B), 100 nM insulin (C), or no unlabeled peptide (D). Lanes E-H: Membranes were labeled with 2 nM 126I-insulin in the presence of no unlabeled peptide (E), 100 nM insulin (F), 100 nM IGF-I (G), or 100 nM IGF-II (H). The receptor-ligand complexes were cross-linked with DSS and electrophoresed on a 5% polyacrylamide gel after treatment with 50 mM dithiothreitol. An autoradiogram of the dried gel is shown. This film has been intentionally overexposed in order to reveal the relatively weak 125I-insulin-labeled species at Mr = 131,000. These data are representative of two replicate experiments.

and IGF-I-binding properties of this species are consistent with the identification of this labeled band as the asubunit of the type I IGF receptor (9, 10). An intense band at 270,000 is also present on the autoradiograms. As both 133,000 and 270,000 species are displaced equipotently by IGF-I, we tentatively conclude that the 270,000 species represents an incomplete reduction product, perhaps an «2-dimer, derived from the type I IGF receptor. This species may also have arisen from intersubunit cross-linking by DSS (24). When 125I-insulin was affinity cross-linked to IEC-6 cell membranes and analyzed by SDS-PAGE, the radiolabel was associated with an Mr = 131,000 band under reducing conditions. However, the intensity of the radiolabeled band was much less than that of the 133,000 band seen with 125I-IGF-I. The 131,000 species may actually represent 125I-insulin cross-linking to the type I IGF receptor, because of the difference in ligand Mr (6,000 for insulin, «7650 for IGF-I). The result of 125Iinsulin binding studies in IEC-6 cell monolayers also showed that specific binding for insulin was too low to allow characterization of the insulin receptor on intact cells. We conclude that IEC-6 cells do not possess specific surface receptors for insulin. Both Scatchard plot and affinity cross-linking analyses revealed that IEC-6 cells have large quantities of the type II IGF receptor. The following data support the conclusion that the 245,000 species represents the type

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IGF RECEPTORS IN AN INTESTINAL EPITHELIAL CELL LINE

II receptor. First, an Mr of 245,000 identified in the present study is typical for the type II receptor in mammalian cells (7, 9, 10, 25). Second, competitive binding and affinity cross-linking studies have demonstrated that recombinant DNA-derived IGF-I has a low affinity for the IEC-6 cell type II receptor, which is consistent with previous results obtained from other types of cells (26, 27). Third, unlabeled insulin was without effect in displacing 125I-IGF-II binding to IEC-6 cells. Fourth, inclusion of mannose-6-phosphate in the incubation medium along with 125I-IGF-II resulted in increased labeling of the 245,000 band. Several studies have demonstrated that mannose-6-phosphate enhances 125I-IGF-II binding to its receptor by 60 -80% in various membrane preparations (16, 18, 28). Fifth, we have tested a polyclonal antibody against the rat type II IGF receptor (29) for its ability to inhibit the binding of 125MGF-II to IEC-6 cell membranes. Affinity cross-linking studies demonstrated that this antibody blocks 125I-IGF-II labeling of the 245,000 species but not 125I-IGF-I labeling of the type I receptor (Park, J. H. Y., and R. G. MacDonald, unpublished observation), confirming that the 245,000 species is indeed the type II IGF receptor. Scatchard analysis of saturation data for 125I-IGF-II binding to IEC-6 cell monolayers revealed curvilinear plots consistent with two classes of binding sites (Fig. 3). This phenomenon made it difficult to measure Bmax and KD in equilibrium binding studies and to assess Ki in competitive inhibition analysis (Fig. 4), particularly since the low affinity sites outnumbered the high affinity sites

by 60 to 1. Thus, the discrepancy between the Ki values calculated for half-maximal inhibition of 125I-IGF-II binding by unlabeled IGF-II (20 nM) and the KD for IGFII binding to the low affinity component (60 nM) may reflect the inaccuracy in estimating these parameters for such a weak binding component. Preliminary experiments suggested that the source of this low affinity binding component may have been the serum in the culture medium rather than the cells. We have observed this same phenomenon in 125I-IGF-II binding experiments using monolayers of H-35 hepatoma cells, and an apparently similar effect was reported by Kiess et al. (30) using BRL-3A2 cells. Furthermore, direct affinity crosslinking analysis failed to reveal any discrete molecular species that might account for this low affinity IGF-II binding, so we cannot definitively attribute this function to IGF-binding proteins. On the other hand, reports by Clemmons and co-workers (31, 32) and De Mellow and Baxter (33) have raised the interesting possibility that IGFs associated with binding proteins at the cell surface may directly modulate IGF-receptor interactions and subsequent biological effects of IGFs. Preliminary studies using 125I-IGF-I ligand blotting have revealed an Mr = 31,000 binding protein present in medium conditioned

3003

by IEC-6 cells (Park, J. H. Y., and R. H. McCusker, unpublished observation). Further experiments on the potential role of this binding protein in IGF action on IEC-6 cells are in progress. Our findings on the IGF and insulin receptors present in IEC-6 cells are generally consistent with previous studies on intestinal epithelium from rat (34) and mouse (35). Laburthe et al. (34) fractionated rat jejunal-ileal epithelium into cell populations corresponding to different regions from the crypt cells to the villus tip. They observed a decreasing gradient of both IGF-I receptors and IGF-II receptors along the crypt-villus axis. In another study, Gallo-Payet and Hugon (35) employed a similar strategy to measure insulin receptors on cells isolated from mouse intestinal epithelium. A gradient of insulin receptor concentration was found that, remarkably, was inverted relative to that of the IGF receptors. IEC-6 cells resemble crypt cells in that they have high levels of both type I and type II IGF receptors and few insulin receptors. A comparison of our data on equilibrium binding of IGF-II to IEC-6 cells with the results of Laburthe et al. (34) on rat intestinal epithelium does reveal several differences. Our studies were done on monolayers of intact IEC-6 cells in order to assess cellsurface receptors that might mediate the proliferative effects of IGFs on these cells. This led to the aforementioned detection of two classes of IGF-II binding sites on IEC-6 cells. However, we have found that the low affinity binding component is lost when membranes are prepared from IEC-6 cells (data not shown). Laburthe et al. (34) employed membrane preparations from intestinal epithelium and found a single class of high affinity IGF-II binding sites. Such differences in methodology may also account for the discrepancy in KD values estimated for IGF-I binding (3.1 nM in our study, 7.2 nM in the previous work (34)) and high affinity IGF-II binding (0.87 nM and 9.5 nM, respectively). These disparate KD estimates for IGF-II binding to the type II receptor in rat intestinal epithelium are, nevertheless, well within the range of values reported for other cells and tissues (6, 14,16, 28). We have previously found that IGFs and insulin stimulate DNA and protein synthesis of IEC-6 cells (see footnote 1). In these experiments, IEC-6 cells were incubated in serum-free medium for 24 h with different concentrations of IGF-I, IGF-II or insulin. Incorporation of [3H]thymidine into DNA and [uC]leucine into protein were stimulated 2-fold by maximally effective concentrations of IGF-I and to a lesser degree by insulin or IGFII (up to 1 fiM). The dose of IGF-I calculated to produce half-maximal stimulation of [3H]thymidine incorporation was 1.6 nM, which is similar to the calculated KD for IGF-I binding to the type I receptor in these cells (Fig. 1). However, these data must be correlated with caution because the measurements were made under very

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IGF RECEPTORS IN AN INTESTINAL EPITHELIAL CELL LINE

Endo • 1990 Vol 126 • No 6

different experimental conditions. Although these results lead to the hypothesis that the type I IGF receptor mediates the proliferative effects of IGF-I in IEC-6 cells, we cannot draw a firm conclusion concerning this question. The complicating factor in this matter is the type II IGF receptor. The primary role of this receptor in transporting lysosomal enzymes from their sites of synthesis in the Golgi to their ultimate destination is well established (25, 36, 37). However, the functional consequences of IGF-II binding to this receptor are still unclear (7,12). Several studies have suggested that the type II receptor may mediate the growth-promoting and metabolic effects of IGF-II in some cell types, but the preponderance of evidence currently favors the view that IGF-II does not initiate a signalling event upon binding

kuhn are surrounded by many fibroblasts (45). Future work will address the hypothesis that fibroblasts underlying intestinal epithelium may produce IGFs which act through a paracrine mechanism to stimulate proliferation of intestinal epithelial cells. In summary, these results indicate that IEC-6 cells have abundant quantities of the type I and II IGF receptors and very few insulin receptors. In conjunction with our previous data on the potent proliferative effect of IGF-I, the present results suggest that the actions of IGFs and insulin are mediated through the type I IGF receptor in IEC-6 cells. IEC-6 cells offer unique opportunities for investigating the mechanism by which these growth factors regulate intestinal epithelial cell growth because of their abundant expression of both types of

to this receptor (7, 12). Of particular importance are the

IGF receptors.

recent observations by Canfield and Kornfeld (38) and Clairmont and Czech (39) that chicken and Xenopus mannose-6-phosphate receptors do not bind IGF-II, despite the fact that at least chickens possess functional IGF-II (40). These findings cast strong doubt upon the hypothesis that the type II receptor is an integral component of the mechanism by which IGF-II elicits biological effects in target cells. Further work will be needed to rigorously establish that the type I receptor mediates the biological actions of the IGFs in IEC-6 cells. The availability of antibodies capable of blocking IGF-I interaction with the rat type I receptor would be particularly useful for this purpose. If IGF-I is an important growth factor in the regulation of intestinal epithelial growth in vivo, it will be of primary interest to determine the source of this IGF-I. The trophic effect of GH on small intestinal mucosa has been well documented (1). However, the mechanism of GH action on the regulation of intestinal epithelium is not known. Data from our laboratory indicate that GH does not directly stimulate IEC-6 cell growth (see footnote 1). Furthermore, IEC-6 cells do not have GH receptors (Park, J. H. Y., and R. G. MacDonald, unpublished observation), suggesting that GH does not have a direct action on intestinal epithelial cells, but possibly an indirect action mediated by another cell type. The growthpromoting effects of GH are generally believed to be mediated through IGFs (5). IGFs, although traditionally considered to originate in the liver, are also synthesized by the fibroblasts of many tissues of the body and may have a paracrine mechanism of action (5). Northern blot and in situ hybridization analyses revealed production of mRNA for both IGF-I and IGF-II in the intestine (4143), and mouse intestinal explants release IGF-I into the surrounding medium (44). These findings raise the possibility that the primary biological actions of IGFs are exerted locally in the intestine near the site of secretion. In the small intestine, the bases of the crypts of Lieber-

Acknowledgments We are grateful to Mrs. M. H. Niedenthal of Lilly Research Laboratories, Indianapolis, IN, for the gifts of porcine insulin and the IGFs. We thank Pamela Walter and Elinor Shanahan for preparation of the manuscript.

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