Demonstration of Receptors for Insulin and Insulin ...

THEJOURNAL OF BIOLOGIC&CHEMISTRY Vol. 257, No. 2, Iesue of January 25, pp. 663-669,1982 Printed in U.S.A.

Demonstration of Receptors for Insulin and Insulin-like Growth Factors on Swarm Rat Chondrosarcoma Chondrocytes EVIDENCE THAT INSULIN STIMULATES PROTEOGLYCAN SYNTHESIS THROUGH THEINSULIN RECEPTOR* (Received for publication, July 29, 1981)

Thomas P.Foley, Jr.$g, S. Peter NissleyS, Richard L. Stevenslgg, George L. Kingll, Vincent C. Hascalll, Rene E. Humbel**, Patricia A. Short$, and Matthew M. RechlerSS From the # Metabolism Branch, National Cancer Institute; 1Laboratory of Biochemistry, National Institute of Dental Research; 11 Diabetes Branch, National Institute of Arthritis, Metabolism, and Digestive Diseases; +# Laboratory of Biochemical Pharmacology, National Institute of Arthritis, Metabolism, and Digestive Diseases, National Znstitutes of Health, Bethesda, Maryland 20205 and the * * Biochemisches Znstitut der Universitat Zurich, Zurich, Switzerland

* A preliminary report of these findings was presented at the62nd Annual Meeting of the Endocrine Society, Washington, D. C., 1980, Abstract 784. The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked“advertisement” in accordance with 18U.S.C. Section 1734 solely to indicate this fact. 8 Supported by the Intergovernment PersonnelAct, National Cancer Institute, National Institutes of Health, during a sabbatical leave of absence from the Department of Pediatrics, Scho-1 of Medicine, University of Pittsburgh, Pittsburgh, PA. Present address, Children’s Hospital of Pittsburgh, 125 DeSoto Street, Pittsburgh, PA 15213. 00 Supported by the Arthritis Foundation, Atlanta, GA. Present address, Department of Medicine, Harvard Medical School and Robert B. Brigham Hospital, Boston, MA 02115.

The somatomedins or insulin-like growth factors’ are potent stimulators of proteoglycan synthesis in cartilage and isolated chondrocyte preparations (1-5). Insulin has also been observed to stimulate proteoglycan synthesis, but the effect requires supraphysiological concentrations whereas the somatomedins are active at physiological concentrations (4, 69). These observations have led to the proposal that the stimulation of proteoglycan synthesis by insulin in the chondrocyte is mediated by a somatomedin receptor rather than an insulin receptor (10, 11). Recently, Stevens and co-workers reported that the rat insulin-like growth factor, MSA? and porcine insulin stimulated proteoglycan synthesis in cultured chrondrocytes derived from the transplantable Swarm rat chondrosarcoma (12, 13). Surprisingly, insulin was found to stimulate [35S]sulfate incorporation with a half-maximal effect at the physiologic concentration of 1ng/ml. The concentration of MSA required for half-maximal stimulation was 100 ng/ml. The [35S]sulfate incorporation data reflected net proteoglycan synthesis since insulin and MSA stimulated the accumulation of hexuronic acid and hexosamine and stimulated the incorporation of r3H] serine, [3H]proline, and [3H]glucosamine into macromolecules. Neither insulin nor MSA stimulated a mitogenic response by the chondrocytes in monolayer culture. The finding that insulin stimulated proteoglycan synthesis at physiologic concentrations suggested that insulin was mediating this response through a classic insulin receptor. Antibodies to the insulin receptor have been employed recently to determine which biologic responses to insulin or MSA are mediated by the insulin receptor. Thus, King et al. (14) showed that the Fab fragment of the anti-insulin receptor antibody blocked the stimulation by insulin and MSA of glucose oxidation in rat adipocytes. Although the antireceptor antibody blocked the binding of ’251-insulinto the insulin receptor, the antibody did not inhibit the binding of “51-MSA to its receptor on the adipocytes. Thus, MSA stimulates glucose oxidation in the ratadipocyte by interacting with the insulin receptor. By contrast, the Fab fragment of the anti-

’ The somatomedin or insulin-like growth factor family includes the following: IGF-I and IGF-11, somatomedin A, somatomedin C, and multiplication-stimulating activity. The abbreviations used are: MSA, multiplication stimulating activity; IGF-I and IGF-11, insulin-like growth factors I and 11; Hepes, 4-(2-hydroxyethyl)-l-piperazineethanesulfonic acid; Tes, N-[tris (hydroxymethyl)methyl-2 amino]ethanesulfonic acid; Bes, 2-[bis(2-hydroxyethyl)amino]ethanesulfonicacid; BSA, bovine serum albumin; IgG, immunoglobulin G.

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The insulin-like growth factor, multiplication stimulating activity (MSA), and insulin were recently shown to stimulate proteoglycan synthesis in monolayer cultures of chondrocytes derived from the Swarm rat chondrosarcoma. Insulin produced significant stimulation at a concentration of less than 1ng/ml, suggesting that insulin was acting through the insulin receptor rather than through a somatomedin receptor. In this paper we have shown that theinsulin-like growth factors IGF-I and IGF-11 also are potent stimulators of [36S]sulfate incorporation into macromolecules recovered from the medium and cell layer matrix of the chondrosarcoma chondrocytes. Proinsulin was 3% as potent as insulin in stimulating [36S]sulfateincorporation. We identified receptors for insulin and the insulinlike growth factors, MSA, IGF-I, and IGF-II. Insulin, at concentrations 1000 times the concentration required to produce the biologic response, did not compete for binding of ‘2SI-MSA-II-1or of ‘261-IGF-IIand only partially competed for ‘261-IGF-Ibinding. Anti-insulin receptor IgG stimulated proteoglycan synthesis and competed for “%insulin binding. Fab fragment prepared from anti-insulin receptor IgG completely blocked the stimulation of [36S]sulfateincorporation into macromolecules byinsulin while onlypartially inhibiting the biologic response to insulin-like growth factors, MSA, IGF-I, and IGF-II. Similarly, the anti-insulin receptor IgG only partially inhibited the binding of‘261-IGF-I and lZ6I-IGF-II while completelyblocking the binding of ’261-insulin.We conclude that insulin stimulates proteoglycan synthesis in the chondrosarcoma chondrocytes by acting through the insulin receptor whereas the insulin-like growth factors probably act through their own receptors.

664

Receptors for Insulin and

IGFChondrosarcoma on Chondrocytes

insulin receptor antibody did not block insulin-stimulated [3H] thymidine incorporation into DNA in human skin fibroblasts; however, the Fab fragment did inhibit binding of 1251-insulin to the human skin fibroblast insulin receptor. Since insulin was known to compete for 1251-MSAbinding to human skin fibroblasts, these experiments implied that the mitogenic response of insulin in these cells was mediated by the MSA receptor. In this report, we describe results which indicate that the stimulation of proteoglycan synthesis in the chondrosarcoma chondrocytes by insulin and somatomedins is mediated through different receptors. EXPERIMENTALPROCEDURES

RESULTS

Stimulation of [35S/SuZfateIncorporation into Proteoglycans of Chondrosarcoma Chondrocytes Treated with Insulin, Proinsulin, and Insulin-like GrowthFactors-Stevens et at. (12) recently reported that insulin and theinsulin-like growth factor, MSA, stimulate proteoglycan synthesis in chondrosarcoma chondrocytes as measured by the incorporation of [35S] sulfate into proteoglycans. We have now tested two other members of the insulin-like growth factor family, IGF-I and IGF-11, for ability to stimulate [35S]sulfate incorporation by these chondrocytes (Fig. 1).As reported by Stevens et al. (12)

/

IGF-I


Materials-Collagenase (Clostridium histolyticun, CLS-11)was obtained from Worthington; trypsin (bovine pancrease) was from Gibco; fetal calf serum, L-glutamine (200 m ~ ) penicillin, , and streptomycin were from Grand Island Biological; Hepes, Tes, Bes, and BSA (A7638) were from Sigma; PD-10 gel filtration columns were from Pharmacia; [%]sulfate and Aquasol were from New England Nuclear; ultrapure guanidine hydrochloride was from Schwarz/Mann. Porcine insulin and proinsulin were gifts from Dr. Ronald Chance, Eli LiUy Co. The other media were prepared by the Media Unit a t the National Institutes of Health. All monolayer cultures were plated on 35-mm Falcon plastic Petri dishes. MSA was purified from the serum-free conditioned medium of a rat liver cell line (BRL-3A) grown in tissue culture (15). The MSA preparations used for most studies (Sephadex (2-75, peak I1 MSA) consisted of four closely migrating MSA species on analytical disc polyacrylamide electrophoresis (pH 2.7, 9 M urea) (15). These four species of M,= 8700 have been shown to have equal biological and immunological specific activities (15, 16). For some experiments, two further purified preparations of MSA (MSA II-1,111-2)were utilized. MSA 11-1and MSA 111-2 were purified by preparative disc polyacrylamide electrophoresis of the Sephadex G-75 peak I1 and peak 111 fractions (15). IGF-I and IGF-I1 were purified to homogeneity from acid-ethanol extracts of human plasma as described earlier (17). The preparation of IGF-I used for these studiescontained 10% IGF-11.1251labeled polypeptides were prepared by previously reported modifications of the chloramine-T method at specific activities of110-160 pCi/pg for insulin (18) and 12-45 pCi/pg for MSA 11-1, IGF-I, and IGF-I1 (19). Antireceptor IgG was obtained from the serum of patients B-2 and B-9 with the type B syndrome of extreme insulin resistance and acanthosis nigricans (20).The intact, bivalent antireceptor IgG preparations and the monovalent Fab fragments were purified as previously described (14).The biological activity of these preparations was confirmed in rat adipocytes by demonstrating the insulinomimetic effect of the bivalent antireceptor IgG and the inhibitory effect of the monovalent Fab fragments on insulin-stimulated glucose oxidation (14).The protein contents of the antireceptor IgG and Fabfragments were determined by the method of Lowry et al. (21) using BSA as standard. Culture of Chondrosarcoma Chondrocytes-The isolation of chondrocytes from the Swarm rat chondrosarcoma tumors and the establishment of primary cultures were performed as previously described (12, 22, 23). Followingtrypsin and collagenase digestion, the isolated chondrocytes were plated in 35-mm dishes at a concentration of 1.0 X lo6 ceUs/ml (1.5 m l ) in Dulbecco's modified Eagle's medium containing 4 gm/liter of glucose and supplemented with 15 mM Hepes, 10 mM Tes, 10 m~ Bes, pH 7.2, 100 units/ml of penicillin, 100 pg/ml of streptomycin (referred to hereafter as DMEM). For '2511"SA or "'IIIGF binding experiments, the chondrocytes were isolated without exposure to serum and plated in serum-free DMEM. The cells were used for binding studies 20 to 48 h later. The serum-free conditions were used to avoid the possibility of '?'II"SA or Iz5I-IGFbinding to specific binding proteins found in serum (24). For 'Z51-insulinbinding studies, the chondrocytes were plated in serum-free DMEM or in DMEM containing 1%fetal calf serum and used for binding studies 20-48 hlater. Chondrocytes that wereused later for[35S]sulfate incorporation studies were plated in serum-free DMEM or DMEM containing either 1 or 10% fetal calf serum. On the day following plating, the medium was replaced with serum-free DMEM and the cultures were starved for 1-2 days before the addition of each growth factor and the determination of [35S]sulfateincorporation. [35S]Sulfate Incorporation into Proteoglycans-After 1-2 days of

starvation in serum-free DMEM at 37 "C in a humidified atmosphere of 5% CO? and 95% air, test materials in DMEM with 0.25% (w/v) BSA were added for 18-20 h. Each medium was replaced with DMEM containing [35S]sulfate(10 pCi/ml) and incubated for 3 h. Incorporation of [35S]sulfateinto macromolecules of both the media and the 4 M guanidine-HC1extracts of the cell matrix layers was determined as previously described (12)using PD-10 columns (prepacked G-25 medium, 9-ml bed volume;Pharmacia) and results were expressed as the sum of the incorporation in medium plus cell matrix. The [%] sulfate incorporated into macromolecular material by chondrocytes is found in proteoglycan. The amounts and types of proteoglycan synthesized by control and insulin-treated chondrosarcoma cells have been well characterized previously (13). Binding of '251-labeledPolypeptides to Chondrosarcoma Chrondrocytes-The cells were isolated and plated as described above. For measurement of the binding of '"1-MSA, IZ51-IGF-I, and '251-IGF-II, each medium was aspirated and the dishes were washed twice with 2 ml of DMEM with 10 mg/ml BSA. A serial dilution of the unlabeled polypeptides in DMEM-BSA together with '251-labeledpolypeptide was added to each cell monolayer in a volume of 0.7 ml. Following 3 h of incubation at 22 "C in 95% air and 5% CO2, each medium was aspirated and the cell layer was washed three times with 2 ml of cold Dulbecco's phosphate-buffered saline and solubilized with 1.0 ml of 1%(w/v) sodium dodecyl sulfate. An aliquot of 0.8 ml was counted in a gamma counter (Beckman 310). For measurement of binding of Iz5Iinsulin, Hepes binding buffer (0.1 M Hepes, 0.12 M NaCl, 1.2 mM MgS04, 2.5 m~ KCl, 10 mM glucose, 1 mM EDTA, 10 mg/ml BSA) (25) was usedin place of DMEM-BSA, and incubations were for 2-3 h at 22 "C in room air.

111-2

1

1

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0.1

1

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10 nglrnl

"100

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1

FIG. 1. Stimulation of [ssS]sulfate incorporation into macromolecules of the media and cell matrix layers of chondrocytes by insulin (0).IGF-I (A), IGF-11 0, MSA 111-2 (0),and MSA 11-1 (A). The net incorporation produced by DMEM containing 10% fetal calf serum (FCS) or by DMEM with 0.25% BSA (control) is shown on the left by the hatched bars.

665

Receptors for Insulin andIGF on Chondrosarcoma Chondrocytes

5 -

-"

-CONTROL-

--- - ---- - -- - -. . . I

1

10

100

-

nglml

FIG. 2. Stimulation of [35S]sulfateincorporation intoproteoglycans of medium and cell matrix by insulin and proinsulin. The level of incorporation produced by DMEM with 0.25% BSA (control) is indicated by the interrupted line.

1

2

3

4

5

6

7

8

9

HOURS

FIG. 3. Time course of '2sII-insulinbinding to the chondrosarcoma chondrocytes in monolayer culture. '251-Insulin(30,600 cpm) in Hepes binding buffer was incubated with monolayer cultures of chondrocytes at 22 "C for the times indicated. Nonspecificbinding was measured in parallel incubations containing excess unlabeled insulin (10pg/ml). After 2 h, buffer in some dishes was replaced with Hepes binding buffer containing 10 pg/ml insulin, dashed line.

changing the medium to medium containing 10 p g / d of unlabeled insulin, only 48% of the radioactivity released into the medium was precipitated by the addition of an equal volume of 20% cold trichloroacetic acid. By comparison, 79% of the 1251-insulin was intact when medium from control dishes (without medium change) was examined for trichloroacetic acid precipitability at 12.5h. These results suggest that although half of the bound '251-insulin dissociated intact from surface binding sites, a portion of the bound '251-insulinhad been degraded at the cell surface or via a pathway involving internalization. The dependence of the specific binding of 1251-insulin on the number of chondrocytes per dish was determined by incubating a series of dishes containing 0.07 to 1.5 X lo6 cells/dish with tracer insulin. There was a linear increase in specific binding of '251-insulin indirect proportion to the increase in cell number (data not shown). Competition for "51-Znsulin Binding by Insulin, IGF-I, IGF-ZZ, MSA IZ-1, and MSA ZZZ-2-The competition by insulin and theinsulin-like growth factors for '251-insulinbinding over the range of concentration of 0.3-10,OOO ng/ml (Table I) demonstrated parallel dose-response curves and a rank order of potency similar to their relative potency in rat adipocytes (11,14) in competing with '251-insulinfor binding to theinsulin receptor or in stimulating glucose oxidation or lipogenesis. Insulin was found to be 20-25 times more potent than IGF-11; IGF-I and MSA-111-2 were 3 times less potent than IGF-11, and MSA 11-1was 6 times less potent than IGF-11. Time Course and Reversibility of Binding of 125Z-MSA-ZI1-Similar to the binding of '251-insulin,the binding of 1251MSA-I1 occurred rapidly at room temperature (Fig. 4). However, in contrast to '251-insulinbinding, the maximum binding of 1251-MSA-II occurred between 4 and 5 and h thengradually decreased. The addition of unlabeled MSA-I1 (2 pg/ml) at 6.5 h resulted in a rapid decline of bound '251-MSA.The amount of intact 1251-MSAreleased into the medium after incubation with unlabeled MSA was determined by trichloroacetic acid precipitation. Seven h afterthe addition of medium containing excess unlabeled MSA, only 48% of the released radioactivity was trichloroacetic acid precipitable. By comparison, 68% of the lZ5I-MSAwas intact when medium from control dishes


under somewhat different culture conditions, insulin in adose as low as 0.5 ng/ml stimulated the incorporation of [35S]sulfate into proteoglycans. The effect of insulin was observed over a 1000-fold concentration range to thehighest dose tested at 0.5 pg/ml. While at the lowest concentrations IGF-I and insulin were equally potent, IGF-I was more potent than insulin at higher concentrations as shown by their divergent, nonparallel dose-response curves. The potency of 10%fetal calf serum was equivalent to 40 ng/ml of IGF-I and 200 ng/ml of insulin. IGF-I1 was approximately 8 times less potent thanIGF-I and the potency of MSA 111-2 (Mr = 7100) and MSA 11-1 (Mr = 8700) were 4 and 8 times less potent than IGF-11, respectively. The dose-response curves for the insulin-like growth factors were nearly parallel to each other but were not parallel to insulin. The ability of insulin to stimulate proteoglycan synthesis at physiologic concentrations of less than 1ng/ml suggested that insulin was acting through the insulin receptor rather than acting as a somatomedin analog through a somatomedin receptor. Proinsulin has usually been reported to be only 3 4 % as potent as insulin in stimulatingavariety of metabolic events which are thought to be mediated throughthe insulin receptor and for competing for binding of '251-insulinto the insulin receptor (26-28). By contrast, proinsulin was 30-50s as potent as insulin in stimulating [3H]thymidine incorporation into DNA and in competing for binding of lZ51-MSAto a somatomedin receptorin chick embryo fibroblasts (29). It was of interest, therefore, to testproinsulin for its ability to stimulate [35S]sulfate incorporation into macromolecules in the chondrosarcoma chondrocytes (Fig. 2). Proinsulin was found to be only 3.2% as potent as insulin. This relative potency of proinsulin to insulin, then, is the same as that observed for the interaction of proinsulin with insulin receptors in insulinresponsive tissues such as the rat adipocyte. Time Course and Reversibilityof Binding of '25Z-Znsulin to Chondrocytes-To examine further the question of whether insulin was stimulating proteoglycan synthesis by interacting with an insulin receptor, we attempted to identify an insulin receptor on these cells. The time course of binding of lZ5Iinsulin to the chondrosarcoma chondrocytes in monolayer culture is shown in Fig. 3. The binding of tracer amounts of insulin occurred rapidly at room temperature, and the level of nonspecific binding as determined by the addition of excess unlabeled insulin was uniformly low. In other experiments, Iz5I-insulinbinding reached a maximum by 10 h that was sustained to 16 h. The addition of unlabeled insulin (10 pg/ ml) at 2 and 10 h (not shown) resulted in a rapid decline of bound '251-insulinto the nonspecific level (Fig. 3). An experiment was done to determine whether the '251-insulinreleased from the cells at 10 h was still intact. Two and one-half h after


666


TABLE I '25Z-Insulinbinding to chondrocytes: competition ZGF-I, ZGF-ZZ, MSA-ZZ-1,and MSA-ZZ-2 Serial dilutions of insulin, IGF-I, IGF-11, MSA-11-1, or MSA-111-2 together with 'Z51-insulin(37,000 cpm) were incubated withmonolayer cultures of chondrocytes for 2 h a t 22 "C in Hepes binding buffer. The concentration of polypeptide required for half-maximal competition is given. Total binding was 3.4% of total radioactivity added; nonspecific binding (in the presence of 10 pg/ml insulin) was 0.5%. b (

Insulin

Concentration (ng/ml)

2.2

Relative potency

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360

180

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1.5

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48

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FIG. 4. Time course of '261-MSA-II-1 binding to chondrosarcoma chondrocytes. Iz5I-MSA(25,000 cpm) in DMEM with 0.25% BSA was incubated with monolayer cultures of the chondrocytes at 22 "C for the times indicated. Nonspecific binding was measured in parallel incubations containing 2 pg/ml unlabeled MSA-11. After 6.5 h,the medium on some dishes was replaced with DMEM-BSA containing 2 pg/ml unlabeled MSA-11, dashed line.

(without medium change) was examined at the time. This result, together with the observation that the amount of specifically bound "'I-MSA declines after 5 h suggest that part of the bound lZ5I-MSAis degraded. There was a linear increase in the specific binding of lZ5IMSA-I1 to the chondrocytes as the cell number increased from 0.01 to 2.2 X lo6 cells/dish (data not shown). The Binding of '25Z-MSA-ZZ-l, '25Z-ZGF-Z, and 'z5Z-ZGF-ZZ to Chondrocytes: Competition by Insulin, MSA-ZZ, IGF-I, and ZGF-ZI-To determine if insulin might stimulate proteoglycan synthesis by interaction with one or more somatomedin receptors, we tested insulin for its ability to compete for binding of radiolabeled MSA-11-1, IGF-I, or IGF-I1 to rat chondrosarcoma chondrocytes in monolayer culture (Fig. 5). Whereas insulin did not compete for binding of either radiolabeled 1251-MSA-IIor "'I-IGF-11, at high concentrations insulin partially competed for binding of 1251-IGF-I. This observation suggests that (a)there are at least two types of somatomedin receptors and ( b )insulin does not mediate its biological effect on proteoglycan synthesis by interacting with the somatomedin receptors. Unlabeled MSA-11, IGF-I, and IGFI1 were tested for their ability to compete for binding of the radiolabeled somatomedins (Fig. 5 ) . The competition curves for binding of 1251-MSA-IIand of "'I-IGF-11 by the somatomedins and insulin were almost identical (Fig. 5 , A and B), suggesting that the MSA-I1 and IGF-I1 receptors are the

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FIG. 5. Binding of '261-MSA-II-1, '261-IGF-II,and lZ51-IGF-Ito chondrosarcoma chondrocytes: competition by unlabeled IGFI, IGF-11, MSA-11, and insulin. Aliquots of '251-MSA-II-1(20,200 cpm), IZ5I-IGF-II(4,570 cpm), or Iz5I-IGF-I(5,200cpm) were incubated with replicate monolayer cultures of chondrocytes togetherwith serial dilutions of each unlabeled polypeptide. Incubation was for 3 h at 22 "C. Maximum specific binding expressed as percentage of radioactivity added was as follows: 'Z51-MSA-II-1,11.2; '251-I-IGF-II,6.25; and 1251-IGF-I, 1.9.Nonspecific binding was: 1251-MSA-II-1, 2.0 Iz5I-IGF11, 0.75; and Iz5I-IGF-I,0.4.


I'

(100)

IGF-I1 IGF-I

same. By contrast, IGF-I was relatively more potent in competing for binding of lZ5I-IGF-I(Fig. 5C) than in competing for binding of 1251-MSA-II-l or '251-IGF-II,and IGF-I1 is more potent than IGF-I in competing for binding of lZ5I-IGF-I(Fig. 5 0 . These results suggest that two types of somatomedin receptors are present on the chondrosarcoma chondrocytes, m e which binds radiolabeled MSA-11-1 and IGF-I1 and does not interact with insulin and a second type which binds lZ5IIGF-I and is insulin sensitive. Comparative Effects of Anti-insulin Receptor ZgG on [35S] sulfate Zncorporation and on '25Z-ZnsuZinBinding-Antibodies that inhibit the binding of 1251-insulinto insulin receptors have been isolated from the seraof patients with an extreme form of insulin resistance (14).The intact, bivalent, antireceptor IgG has been shown to mimic many of the biological effects of insulin in vitro (30-35). Therefore, we examined the effect of an anti-insulin receptor IgG preparation on "'I-insulin binding and [35S]sulfateincorporation in the chondrosarcoma chondrocytes. The addition of increasing concentrations of the bivalent anti-insulin receptor IgG to thechondrocyte monolayer cultures stimulated the net incorporation of [35S]sulfateinto proteoglycans (Fig. 6a). Thelack of an insulinomimetic response by IgG purified from the serum of control patients and the linear dose-response curve for the anti-insulin receptor IgG parallel to that for insulin suggest that the bivalent anti-insulin receptor IgG mimics insulin action in the chondrosarcoma chondrocytes by interacting with the insulin receptor. The specificity of this biological effect is supported by the competition for binding of lz5I-

Receptors for Insulin and IGFChondrosarcoma on Chondrocytes insulin by the bivalent anti-insulin receptor IgG, but not by IgG from control patients (Fig. 6b). Furthermore, the effects of the anti-insulin receptor IgG in the stimulation of [35S] sulfate incorporation and in the competition for 1251-insulin binding were achieved over the same concentration range of IgG. The Effects ofAnti-insulin Receptor IgGFragment Fab on Stimulation off35S]Sulfate Incorporation by Insulin, MSA11,IGF-I, andIGF-11-In contrast to the intact anti-insulin receptor IgG which mimics the biological effects of insulin, the monovalent Fab fragment preparation is without biologic effect and hasbeen shown to block insulin action in vitro (14,

667

35). Therefore, the anti-insulin receptor Fab fragment was tested for its ability to inhibit the stimulation of [35S]sulfate incorporation by insulin and insulin-like growth factors. The addition of 5 pg/ml anti-insulin receptor fragment Fab to chondrocytes incubated with concentrations of insulin from 1-10 ng/ml caused partial inhibition of insulin stimulated [35S]

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FIG. 6. Stimulation of [36S]sulfateincorporation into macromolecules and competition for '251-insulinbinding by antiinsulin receptor IgG. a, monolayer cultures of chondrocytes were

FIG. 7. Effect of Fab fragment prepared from the anti-insuIin receptor IgG on stimulation of [35S]sulfateincorporation into proteoglycans by insulin and insulin-like growth factors. incubated with DMEM-BSA containing either insulin, anti-insulin Increasing concentrations of insulin (a),MSA ( b ) ,IGF-I ( c ) , or IGFreceptor IgG (patient B-9), or control IgG. Incorporation of [35S] I1 ( d ) were incubated with chondrocytes either alone (filled circles) sulfate into macromolecules (media plus cell layer matrix extracts) was measured as described under "Experimental Procedures." b, aliquots of '251-insulin(27,200 cpm) were added to monolayer cultures together with serial dilutions of either insulin, anti-insulin receptor IgG (patient B-9), or control IgG. Incubation was for 3 h at 22 "C.

or together with Fab fragment (5 pg/ml) prepared from anti-insulin receptor IgG (patient B-2) (open circles) or from control IgG (open triangles). [?S]Sulfate incorporation was determined as described under "Experimental Procedures." The level of incorporation for DMEM-BSA alone is indicated by the hatched bars.

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FIG. 8. Effect of Fab fragmentprepared from the anti-insulin receptor IgG on stimulation of [35S]sulfateincorporationinto proteoglycans by insulin and insulin-like growth factors. The experiment was the same as in Fig. 7 except the concentration of Fab fragment was 10 pg/ml; control Fab was omitted from the experiments in 6-d.


8

Receptors for Insulin and IGFChondrosarcoma on Chondrocytes

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sulfate incorporation into proteoglycans (Fig. 7a),shifting the dose-response curve approximately 4-fold to the right. No inhibition was observed when purified IgG fragment Fabfrom control patients was added. There was no significant inhibitory effect on anti-insulin receptor fragment Fab onthe stimulation of [35S]sulfate incorporation by MSA-11, IGF-I, or IGF-I1 (Fig. 7, b-d). When a higher concentration of anti-insulin receptor fragment Fab (10 pg/ml) was used in a similar experiment (Fig. 8). the stimulation of [35S]sulfateincorporation by insulin was completely inhibited (Fig. 8a),while stimulation by either MSA-11, IGF-I, or IGF-I1 was now partially inhibited (Fig. 8, b-d) . In the concentration range of 1-100 pg/ml of anti-insulin receptor IgG, there was no inhibition of the binding of lZ5IMSA-I1 to the chondrocytes and only minimal inhibition of the binding of Iz5I-IGF-Iand '"1-IGF-11 (Fig. 9). Thus, the relatively greater effectiveness of the anti-receptor Fab fragment in inhibiting the biological response of the chondrocyte to insulin compared to its inhibition of tl. response to the insulin-like growth factors is reflected by the relative potency of the IgG preparation from the same patientin inhibiting the binding of radiolabeled insulin and insulin-like growth factors (Fig. 9). DISCUSSION

The finding by Stevens et al. (12) that physiologic concentrations of insulin stimulated in vitro proteoglycan synthesis in the Swarm rat chondrosarcoma chondrocyte led to the proposal that this response to insulin was mediated by the insulin receptor. This proposal was contrary to thehypothesis that the effect of insulin on [35S]sulfateincorporation in normal cartilage tissue and isolated chondrocytes was a result of insulin acting through a somatomedin receptor (10, 11). In this study, we have shown the following additional evidence in support of insulin acting through an insulin receptor to stimulate proteoglycan synthesis in these chondrocytes. (a) Proinsulin was only 3% as potent as insulin in stimulating [35S]sulfateincorporation into macromolecules. This relative potency of proinsulin to insulin IS typical for interaction with an insulin receptor (26-28) and is in contrast to the behavior


FIG. 9. Competition by anti-insulin receptor IgG for binding of '*'I-insulin, '2sI-IGF-I, '*'I-IGF-II, or '251-MSA-II-1 to the chondrosarcoma chondrocytes. Increasing concentrations of antiinsulin receptor IgG (patient B-2) were added to '251-insulin(23,650 cpm), Iz5I-IGF-I(5,200 cprn), '251-IGF-II(4,570 cpm), or '251-MSA-III (20,200 cpm) and incubated with replicate monolayer cultures of chondrocytes for 3 h at 22 "C. Maximum specific binding expressed as percentage of radioactivity was as follows: '251-insulin,2.1; ' 2 5 ~ - ~ I, 1.9; '251-IGF-II,6.25; and 1251-MSA-II-1,11.2. Nonspecific binding was: 'Z51-insulin, 1.3; lZ5I-IGF-I,0.4; '251-IGF-II,0.75; and '251-MSA-II1, 2.0.

of proinsulin in interacting with a somatomedin receptor where proinsulin was found to be 30-50% as potent as insulin (29). ( b ) Insulin competed with '251-insulinfor binding to the chondrocytes over the same concentrationrange required for stimulation of ["%]sulfate incorporation. ( c ) A naturally occurring anti-insulin receptor IgG preparation stimulated [35S] sulfate incorporation and competed for 1251-insulin binding to an insulin receptor on the chondrocytes. Anti-insulin receptor IgG preparations previously have been shown to mimic a variety of insulin-stimulated metabolic actions in vitro (3035). The observation that anti-insulin receptor IgG stimulates [35S]sulfateincorporation into macromolecules in the chondrosarcoma chondrocyte points to the insulin receptor as an important component in the sequence of events leading to the biologic response. (d)A monovalent Fab fragment prepared from the anti-insulin receptor IgG completely inhibited insulin stimulated [35S]sulfateincorporation while only partially inhibiting the stimulation by the insulin-like growth factors. The anti-insulin receptor Fab fragment had been shown previously to block the metabolic effects of insulin in vitro (14). ( e ) Insulin did not interact with insulin-like growth factor ~ receptors ~ at concentrations required for producing the biologic response, suggesting that insulin does not stimulate [35S]su1fate incorporation by interacting with these receptors. Although we have identified receptors for the insulin-like growth factors, MSA, IGF-I, and IGF-11, on the chondrosarcoma chondrocytes, the evidence that thebiologic response to these polypeptides is the result of interaction with insulin-like growth factor receptors is not as conclusive as the evidence for insulin acting through an insulin receptor. Our data suggest that there are two types of insulin-like growth factor receptors on the chondrosarcoma chondrocytes. When the binding of 1251-IGF-II and lZ5I-MSAwas examined, insulin did not compete for binding and the rank order of potency of IGF-11, MSA, and IGF-Iwas identical in competing for binding of the two radioligands. However, when lZ5I-IGF-Iwas used as radioligand, IGF-I was found to be more potent than it was in competing for the binding of '"1-IGF-11 or of Iz5I-MSA, and insulin competed partially for binding. These findings are similar to the binding data observed for a rat liver cell line (BRL-3A2) and for purified rat liver membranes (36) where two types of insulin-like growth factor receptors were identified, one that preferred IGF-I1 and MSA and was not recognizedby insulin and a second which preferred IGF-I and interacted with insulin. It is difficult to explain the findings that IGF-I was more potent than IGF-I1 in stimulatingthe biologic response, while it was less potent than IGF-I1 in competing for binding of Iz5IIGF-I, 'ZSI-IGF-II,and '"I-MSA, if indeed the biologic response to the insulin-like growth factors is mediated by IGF receptors. A possible explanation for this discrepancy is that although there is a receptor which prefers IGF-I, the binding capacity of this receptor is so low compared to thecapacity of the IGF-II/MSA receptor that the dose-response curves of IGF-I, IGF-11, and MSA for competition for Iz5I-IGF-Ibinding reflect the pattern for the IGF-II/MSA receptor. Since we have not examined the time course of the biologic response to IGF-I andIGF-11, it is also possible that, at20 h after addition of the polypeptides to the starved chondrocytes, the biological response to IGF-I has already reached a plateau while the response to IGF-I1 is still increasing. A high concentration of the anti-insulin receptor Fab fragment was found to partially inhibit the biological response to the insulin-like growth factors. One explanation of this finding is that partof the biologic response to theinsulin-like growth factors might be mediated through the insulin receptor. However, high concentrations of the anti-insulin receptor IgG



Acknowledgments-We thank Maxine Lesniak and her colleagues in the Diabetes Branch, National Institute of Arthritis, Metabolism and Digestive Diseases, for providing '251-insulin. Wealso thank Dr. James Kimura, National Institute of Dental Research, for discussions and help in culturing the chondrosarcoma chondrocytes. REFERENCES 1 Froesch, E. R., Zapf, J., Audhya, T. K., Ben-Porath, E., Segen, B.

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preparation also inhibited the binding of '251-IGF-Iand lZ5IIGF-I1 to their receptors, suggesting that inhibition by the anti-insulin receptor Fab fragment was a result of partial blockade of the insulin-like growth factor receptors. It is not known whether this interaction of the anti-insulin receptor antibody with the insulin-like growth factor receptors is due to the presence of naturally occurring anti-IGF receptor antibodies in addition to the anti-insulin receptor antibodies or rather is a result of antigenic similarities between the insulin receptor and the insulin-like growth factor receptors. Why does insulinstimulate proteoglycan synthesis at physiologic concentrations in the Swarm rat chondrosarcoma chondrocyte by interacting with an insulin receptor whereas pharmacologic concentrations of insulin are required to stimulate the same biologic response in cartilage tissue preparations and isolated chondrocytes, presumably through a somatomedin receptor? Since the chondrosarcoma chondrocyte makes a fetal type proteoglycan, these chondrocytes may resemble embryonic cartilage (12). Perhaps the response to insulin is related to this embryonic character. Alternatively, as proposed earlier (12), the increased sensitivity to insulin may be a property of the transformed phenotype. Malignant mouse mammary cells show a greater sensitivity to insulin with respect to stimulation of growth than untransformed mammary cells (37). Physiologic concentrations of insulin support the growth of a rat liver cell line derived from a hepatoma (38) and the multiplication of the embryonal carcinoma cell l i e F9(39). McCumbee and Lebovitz (40) and Salomon et al. (41) have providedevidence that the Swarm rat chondrosarcoma is under the control of both insulin and insulin-like growth factors in uiuo. Our findings suggestthat atleast a part of this control (proteoglycan synthesis) is mediated through separate receptors for insulin and the insulin-like growth factors. The chondrosarcoma chondrocyte inmonolayer culture should provide an excellent system in whichto study the postreceptor events in the pathways leading to proteoglycan synthesis.

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Demonstration of receptors for insulin and insulin-like growth factors on Swarm rat chondrosarcoma chondrocytes. Evidence that insulin stimulates proteoglycan synthesis through the insulin receptor. T P Foley, Jr, S P Nissley, R L Stevens, G L King, V C Hascall, R E Humbel, P A Short and M M Rechler J. Biol. Chem. 1982, 257:663-669.

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