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insulin hybrid receptors in human endothelial cells
Molecular and Cellular Endocrinology 229 (2005) 31–37
IGF-I/insulin hybrid receptors in human endothelial cells Marloes Dekker Niterta , Simona I. Chisalitaa , Karolina Olssona , Karin E. Bornfeldtb , Hans J. Arnqvista,∗ a
Diabetes Research Center and Division of Cell Biology, Department of Biomedicine and Surgery, Faculty of Health Sciences, Link¨oping University, S-581 85 Link¨oping, Sweden b Department of Pathology, P.O. Box 357470, University of Washington School of Medicine, Seattle, WA 98195-7470, USA Received 23 June 2004; received in revised form 7 October 2004; accepted 12 October 2004
Abstract Vascular complications are common in diabetes. IGF-I receptors (IGF-IR) and insulin receptors (IR) in endothelial cells might respond to altered levels of IGF-I and insulin, resulting in altered endothelial function in diabetes. We therefore studied IGF-IR and IR gene expression, ligand binding, receptor protein, and phosphorylation in human umbilical vein endothelial cells (HUVEC). IGF-IR mRNA was more abundant than IR mRNA in freshly isolated HUVEC (IGF-IR/IR ratio 7.1 ± 1.5) and in cultured HUVEC (ratio 3.5 ± 0.51). Accordingly, specific binding of 125 I-IGF-I (0.64 ± 0.25%) was higher than that of 125 I-insulin (0.25 ± 0.09%). Protein was detected for both receptors and IGF-I/insulin hybrid receptors. IGF-IR phosphorylation was stimulated by 10−10 to 10−8 M IGF-I. IR were activated by 10−9 to 10−8 M insulin and IGF-I. We conclude that HUVEC express more IGF-IR than IR, and also express hybrid receptors. Both IGF-I and insulin phosphorylate their own receptors but only IGF-I seems to phosphorylate hybrid receptors. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: IGF-I receptor; Insulin receptor; Insulin/IGF-I hybrid receptor; Endothelial cells; Human
1. Introduction The endothelium is a dynamic tissue which plays a major role in the regulation of vascular tone; it also has a function in haemostasis, permeability and inflammation. Endothelial dysfunction is considered to play an important role in the development of atherosclerosis (Cines et al., 1998; Hsueh and Quinones, 2003). Hyperinsulinemia, insulin resistance as well as altered IGF-I levels have been implicated in the pathogenesis of cardiovascular disease (Dandona et al., 2003; Juul et al., 2002), although the mechanisms are not clear. Moreover, the presence and function of IGF-I receptors (IGF-IR) and insulin receptors (IR) in endothelial cells are not well characterised. The IGF-IR and IR are homologous tetrameric receptors, with two heterodimers consisting of an extracellular ␣-subunit and a transmembrane -subunit. The ␣-subunit ∗
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0303-7207/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2004.10.003
contains the ligand-binding site; the -subunit has the ligandsensitive tyrosine kinase site that is trans-autophosphorylated after ligand binding (Treadway et al., 1991; Frattali et al., 1992). Both IGF-I and insulin bind to either receptor, but the affinity for their cognate receptor is 100–1000 times higher. Hybrid IGF-I/insulin receptors with one IGF-IR heterodimer and one IR heterodimer have also been found, particularly in tissues where IGF-IR numbers are significantly different from IR numbers (Bailyes et al., 1997; Federici et al., 1997). Hybrid IGF-I/insulin receptors are reported to behave in a manner similar to IGF-IR with respect to ligand-induced autophosphorylation, receptor internalisation, and degradation (Seely et al., 1995). The presence of hybrid IGF-I/insulin receptors and their proportion to IGF-IR and IR could influence the cellular effects of IGF-I and insulin. We investigated the presence of IGF-IR, IR, and hybrid IGF-I/insulin receptors in human umbilical vein endothelial cells (HUVEC). We also studied whether IGF-I and insulin could stimulate tyrosine phosphorylation of the receptors.
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2. Materials and methods
2.3. Real time RT-PCR analysis
2.1. Materials
The freshly isolated or cultured cells were washed with PBS and total RNA was harvested with the Qiagen RNAeasy mini kit (Qiagen GmbH, Hilden, Germany). One micrograms of RNA was reverse transcribed with random primers and Superscript II Rnase H reverse transcriptase. The cDNA was stored in aliquots at −20 ◦ C until use. Comparative quantitative PCR analysis was performed with primer/probe pairs for IGF-I, insulin receptor and IGFI receptor mRNA. The reaction consisted of 25 ng cDNA, 50 nM probe, 300 nM of sense and anti-sense primers, 12.5 l 2× TaqMan mastermix (PE Applied Biosystem, Stockholm, Sweden), and water in a total volume of 25 l. The following primers and probes were used: IGF-I receptor forward primer: 5 -TTT CAA CAA GCC CAC AGG GT-3 , IGF-I receptor reverse primer: 5 -CCA CGA TGC CTG TCT CAC G-3 , IGF-I receptor probe: 5 (FAM)-TGG CTC CAG CAG TCG GAG GGC (TAMRA)-3 ; insulin receptor forward primer: 5 -AGG AGC CCA ATG GTC TGA-3 , insulin receptor reverse primer: 5 -CAG ACG CAG AGA TGC AGC-3 and insulin receptor probe: 5 (FAM)-ACC ATA TCG CCG ATA ACT CAC TTC ATA CAG (TAMRA)-3 . The PCR program started with an initial temperature of 50 ◦ C for 2 min and increased to 95 ◦ C for 10 min, followed by 40 amplification cycles of 15 s at 95 ◦ C and 1 min at 60 ◦ C. An ABI Prism 7700 sequence detector equipped with a 96-well thermal cycler connected to Sequence Detector v1.6.3 software (Applied Biosystems, Stockholm, Sweden) was used in the experiments. The relative IGF-I, insulin receptor and IGF-I receptor mRNA amount was normalised to the housekeeping gene encoding GAPDH. GAPDH reactions of all samples were run in the same 96-well plate in the same run as the reactions of the samples for the genes of interest. 1.25 l GAPDH mastermix (PE Biosystem) consisting of primers and probe for GAPDH was added to 25 ng of cDNA, 12.5 l TaqMan mastermix and water for a total reaction volume of 25 l. Reactions were performed in duplicate. The relative quantity of, insulin receptor and IGF-I receptor mRNA was calculated using the comparative CT method after initial experiments showed similar quantitative PCR efficiency rates for IR, IGF-IR and GAPDH.
Primers and probes were purchased from SGS (Scandinavian Gene Synthesis AB, K¨oping, Sweden). Mono-125 I(Tyr A14)-human insulin (1500 Ci/mmol) and 125 I-IGF-I (2000 Ci/mmol) were obtained from Amersham Pharmacia Biotechnology (Buckinghamshire, UK). The following antibodies were obtained from Santa Cruz biotechnology (Santa Cruz, CA): Polyclonal anti-IGF-IR -subunit antibodies (C20, sc-713) and polyclonal anti-IR -subunit antibodies (C19, sc-711) used for immunoprecipitation and immunoblot. A monoclonal anti-IGF-IR ␣-subunit antibody (2C8, sc-463) and a monoclonal anti-IR -subunit antibody (29B4, sc-09) used for immunoprecipitation, and an anti-phosphotyrosine antibody (PY20, sc-508) used for immunoblot. The monoclonal anti-IR ␣-subunit 83-7 antibody and monoclonal anti-IGF-IR 17-69 antibody were kind gifts of Prof. K. Siddle (Cambridge University, Cambridge, UK). Goat anti-rabbit-HRP (Zymed, San Francisco, CA), sheep anti-mouse HRP (Amersham, Bucks, UK) and streptavidinHRP (Amersham, Bucks, UK) were used as secondary antibodies. Enhance chemiluminescence detection system was obtained from Amersham Pharmacia Biotechnology (Bucks, UK).
2.2. Cell culture EC were isolated from human umbilical veins by a 15 min collagenase digestion (0.5 mg/ml DMEM containing 25 mM HEPES). Briefly, the umbilical veins were collected in a sterile container with PBS and penicillin (100 IU/ml), streptomycin (100 IU/ml) and gentamycin (10 g/ml) at 4 ◦ C. Each umbilical vein was washed with sterile PBS and then treated for 15 min with 100 mg collagenase H in medium A (200 ml DMEM containing 5 ml HEPES and 10 IU/ml penicillin and 10 IU/ml streptomycin) at 37 ◦ C. The cells were collected in 10 ml medium A and the veins were washed with sterile PBS and the flow-through was also collected. The cells were centrifuged at 500 × g for 5 min, washed with culture medium and plated out in culture flasks coated with 0.2% gelatine. Freshly isolated cells were collected for experiments after the second centrifugation. The cells were cultured in DMEM supplemented with 8% heat-inactivated foetal calf serum (FCS) and EGM-2 singlequots® (Bio-Whittaker, Cambrex, Walkersville, MD, USA) with 0.04% hydrocortisone, 0.04% human fibroblast growth factor, 0.1% vascular endothelial growth factor, 0.1% R3 -IGF-I, 0.1% ascorbic acid, 0.1% human epithelial growth factor, 0.1% gentamycinamphotericin, 2 g/ml fungizone, and 0.1% heparin was also added. Cultured cells were used between passages 1 and 2. Cultured control cells were subjected to DMEM medium containing 2% FCS and fungizone for 24 h prior to harvesting of the cells.
2.4. Binding assays HUVEC were cultured to near-confluence in six-well plates and incubated for 4 h at 10 ◦ C in HEPES binding buffer, pH 7.8, of the following composition (mmol/l): HEPES 100, NaCl 120, KCl 5, MgSO4 1.2, glucose 8 and 0.1% bovine serum albumin with the addition of 125 I-IGF-I (12.5 × 10−12 M) or 125 I-insulin (43.9 × 10−12 M) and unlabeled polypeptides at indicated concentrations. The cells were then washed 4 times with ice-cold PBS buffer and dissolved in 0.1% SDS and the radioactivity was measured in a gamma counter. Unspecific binding was defined as binding
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of 125 I-IGF-I or 125 I-insulin in the presence of 10−7 M unlabeled IGF-I or insulin. The binding data were analysed using GraphPad Prism program (GraphPad Software Inc., San Diego, USA) and are expressed either as per cent of the total 125 I-polypeptide bound (in displacement curves) or as percent specific binding of total 125 I-polypeptide added to the medium.
2.5. Immunoprecipitation and Western blot analyses Stimulation of receptor tyrosine kinase phosphorylation was studied in confluent HUVEC that were exposed to lowserum (2%) DMEM for 18 h. The cells were washed with fresh low-serum medium and incubated with 50 M Na3 VO4 in low-serum medium containing 1 mg/ml BSA for 30 min on ice. The cells were then stimulated with insulin or IGF-I in pre-warmed low-serum medium at indicated concentrations or subjected to pre-warmed low-serum medium only for 10 min at 37 ◦ C .1.5 ml lysis buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% sodiumdeoxycholate and 0.5% Triton X-100 supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mg/ml leupeptin, 200 mM Na3 VO4 , 1 mg/ml aprotinin) was added to the cells and incubated for 30 min on ice. Cell lysates were then harvested, centrifuged at 4 ◦ C at 20,000 × g for 15 min and stored at −70 ◦ C until use. The protein content of the samples was determined with the bicinchoninic acid method (Pierce, Rockford, IL, USA). For immunoprecipitation experiments, 0.5 ml cell lysate (containing approximately 500 g of protein) was incubated with 1.25 l immunoprecipitating antibody bound to protein A sepharose for the polyclonal rabbit antibodies or to protein G sepharose for the monoclonal mouse antibodies. The immunoprecipitates were collected, washed and diluted in 25 l of 2× Laemmli sample buffer. The immunoprecipitated samples were boiled for 3 min, centrifuged and separated on a 7.5% SDS–PAGE (Bio-Rad). The samples were electrotransferred onto a PVDF membrane and unspecific antibody binding was blocked by a 60 min-incubation of the membrane with 5% non-fat dry milk in TBS-0.1% Tween 20. The membrane was incubated overnight at 4 ◦ C with primary antibody, followed by a 60 min incubation with secondary antibody. Detection of the protein was performed by enhanced chemiluminescence. Between the steps the membrane was washed with TBS-0.1% Tween 20 wash buffer. The antibodies were diluted in wash buffer. Secondary antibody dilutions contained 2.5% non-fat dry milk except the sheep anti-mouse IgG which was diluted in wash buffer containing 3% bovine serum albumin. Preliminary experiments showed that the supernatant of the cell lysate after immunoprecipitation still contained IGFIR and IR in amounts that could be immunoprecipitated and blotted (results not shown). Even after a third immunoprecipition with antibodies against IGF-IR and/or IR, the receptors could be shown on immunoblot. We have therefore used
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supernatants of cell lysates, that were previously immunoprecipitated with monoclonal IGF-IR 2C8 or monoclonal IR 29B4, for immunoprecipitation with polyclonal IGF-IR C20 and IR C19 in order to show phosphorylation of the tyrosine kinase of the receptors. After successful blotting with the first primary antibody, the membrane was stripped of antibodies by a 30 min incubation in stripping buffer consisting of 10% SDS, 100 mM S-mercaptoethanol, 62.5 mM Tris–HCl (pH 6.7) at 55 ◦ C. In order to detect hybrid receptor, the membrane was subsequentially blotted with primary antibody against the other receptor. 2.6. Statistical analysis Values are given as mean ± S.E.M. Potential differences were analysed with Student’s t-test with P < 0.05 considered significant.
3. Results 3.1. Gene expression Detectable levels of mRNA for IGF-IR and IR were demonstrated in total RNA obtained from HUVEC cultured for two passages (Fig. 1). IGF-IR mRNA was more abundantly expressed than IR mRNA. After standardization of the amount of receptor mRNA to the amount of the housekeeping gene GAPDH mRNA, it was found that cultured HUVEC contained approximately 3.5 ± 0.51 (P < 0.001) times more IGF-IR mRNA than IR mRNA. IGF-IR mRNA and IR mRNA were also detected in total RNA obtained from freshly isolated HUVEC (n = 6). Freshly isolated HUVEC contained more IGF-IR mRNA than IR mRNA; 7.1 ± 1.5 (P < 0.001) fold more.
Fig. 1. Expression of IGF-IR and IR mRNA in cultured HUVEC. Results are presented as relative mean amount of mRNA relative to the mean amount of IR mRNA ± S.E.M. (n = 6 for each).
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Fig. 2. Displacement of 125 I-labelled IGF-I from its receptor by addition of increasing concentrations of unlabeled IGF-I or insulin. Near-confluent cells were incubated for 4 h at 10 ◦ C in Hepes buffer (mean ± S.E.M., n = 4).
3.2. Ligand binding The specific binding of 125 I-IGF-I (0.64 ± 0.25%, mean ± S.E.M.) was higher in HUVEC than the specific binding of 125 I-insulin (0.25 ± 0.092%, mean ± S.E.M.). This indicates the presence of a larger amount of IGF-IR than IR on the plasma membrane of HUVEC. Competition studies with unlabeled polypeptide (n = 4) showed a more potent displacement of 125 I-IGF-I with unlabeled IGF-I than with unlabeled insulin (Fig. 2). The IC50 value, indicating the concentration needed to give halfmaximal displacement, was approximately 200-fold lower for IGF-I (3.6 × 10−10 M, confidence interval 3.2 × 10−11 to 4.04 × 10−9 M) than for insulin (8.25 × 10−8 M, confidence interval 3.28 × 10−9 to 2.08 × 10−7 M). The difference was statistically significant (P < 0.0001). Conversely, 125 I-insulin was more effectively displaced by unlabeled insulin than unlabeled IGF-I (Fig. 3). The IC50 value for 125 Iinsulin displacement by insulin was 2.60 × 10−10 M (confidence interval 4.50 × 10−11 to 1.47 × 10−9 M) and for IGFI was 7.39 × 10−9 M (confidence interval 8.30 × 10−10 to 6.5 × 10−8 M), a 30-fold difference in potency (P < 0.0001). Western blot analysis of receptor protein expression and activation.
Fig. 3. Displacement of 125 I-labelled insulin from its receptor by addition of increasing concentrations of unlabeled insulin or IGF-I. Near-confluent cells were incubated for 4 h at 10 ◦ C in Hepes buffer (mean ± S.E.M., n = 4).
Fig. 4. Tyrosine phosphorylation of IGF-IR -subunit and protein expression of IGF-IR -subunit and IR -subunit after immunoprecipitation with IGF-IR C20 antibody. Cells were stimulated with IGF-I or insulin for 10 min at indicated concentrations. Immunoblot with phosphotyrosine PY20 antibody (upper panel), IGF-IR C20 antibody (middle panel) and IR C19 antibody (lower panel). Results are representative of three experiments. M: molecular weight marker; CTL: control; Ins: insulin; the arrow indicates the molecular weight marker at 97 kDa.
IGF-IR protein could be detected in cell lysates from cultured HUVEC after immunoprecipitation with a polyclonal antibody against the IGF-IR -subunit (C20) followed by immunoblotting with the same antibody (Fig. 4, middle panel). IR protein was also found in HUVEC cell lysate after immunoprecipitation and immunoblotting with a polyclonal antibody against the IR -subunit (C19) (Fig. 5, middle panel). The -subunit of the IGF-IR is slightly larger (97 kDa) than that of the IR (95 kDa) and this difference was reflected in the location of the immunoreactive bands on the blot. These results were confirmed after immunoprecipitation with either a monoclonal anti-IGF-IR (2C8) antibody or a monoclonal anti-IR (29B4) antibody followed by immunoblotting with polyclonal antibodies (results not shown). Stimulation of IGF-IR phosphorylation was studied after immunoprecipitation with anti-IGF-IR (C20) and immunoblot with anti-phosphotyrosine (PY20) antibodies. The IGF-IR tyrosine kinase was activated by IGF-I at concentrations ranging from 10−10 to 10−8 M (Fig. 4 upper panel, results of 10−8 M not shown). The IGF-IR did not show phosphorylation after stimulation with the same concentrations
Fig. 5. Tyrosine phosphorylation of IR -subunit and protein expression of IR -subunit and IGF-IR -subunit after immunoprecipitation with IR C19 antibody. Cells were stimulated with insulin or IGF-I for 10 min at indicated concentrations. Immunoblot with phosphotyrosine PY20 antibody (upper panel), IR C19 antibody (middle panel) and IGF-IR C20 antibody (lower panel). Results are representative of three experiments. M: molecular weight marker; CTL: control; Ins: insulin.
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photyrosine immunoreactive bands of IR after stimulation with IGF-I (Fig. 5 upper panel) could represent phosphorylation of the IR -subunit as well as the IGF-IR -subunit of IGF-I/insulin hybrid receptors.
4. Discussion
Fig. 6. Hybrid receptor protein in HUVEC after immunoprecipitation with monoclonal antibodies. Cells were stimulated with insulin or IGF-I for 10 min at indicated concentrations. (A) Immunoprecipitation with IGF-IR 17-69 antibody and immunoblot with IGF-IR C20 antibody (upper panel) or IR C19 antibody (lower panel). (B) Immunoprecipitation with IR 83-7 antibody and immunoblot with IR C19 antibody (upper panel) or IGF-IR C20 antibody (lower panel). Immunoreactive bands are located at different locations on the blot. Results are representative of three experiments. M: molecular weight marker; CTL: control; Ins: insulin.
of insulin. Stimulation of IR tyrosine kinase phosphorylation was determined after immunoprecipitation with anti-IR (C19) and immunoblot with anti-phosphotyrosine (PY20) antibodies. The IR was phosphorylated after stimulation with 10−9 to 10−8 M insulin but not convincingly activated after stimulation with 10−10 M insulin. IGF-I (10−9 and 10−8 M) was also able to cause a phosphorylation of the IR (Fig. 5, upper panel). The lower part of the phosphotyrosine immunoreactive bands in the IGF-I stimulated cells was located at the same level in the blot as the bands of the cells stimulated with insulin. After stimulation with IGF-I, however, the resulting band was broader and more diffuse in the upper margin. When the lysates were immunoprecipitated with polyclonal IGF-IR -subunit antibodies (C20) and subsequently immunoblotted with polyclonal IR -subunit antibodies (C19), a band could be detected on the blot (Fig. 4, lower panel). This band corresponded to the lower molecular weight band of the -subunit of the IR (95 kDa). Immunoprecipitation with polyclonal IR antibodies (C19) and blotting with the polyclonal IGF-I receptor antibody (C20) revealed the presence of a 97 kDa band which matched the molecular weight of the -subunit of the IGF-IR (Fig. 5, lower panel). Identical results were obtained by immunoprecipitating cell lysates with monoclonal antibodies for IGF-IR (17-69) or IR (837), the subsequent immunoblot with polyclonal antibodies against the -subunit of either receptor (C19 or C20) showed the presence of two distinct bands corresponding to 97 and 95 kDa (Fig. 6). The presence of two distinct immunoreactive bands is an indication for the presence of hybrid IGF-I/insulin receptors on HUVEC plasma membrane. The diffuse phos-
In this study, HUVEC were found to express IGF-IR and IR as assessed with gene expression, ligand binding and Western blot. We found higher expression of IGF-IR than IR. A novel finding is the presence of hybrid IGF-I/insulin receptors in human vascular endothelial cells. Both IGF-I and insulin phosphorylate their own receptors but only IGF-I seems to activate hybrid receptors. IGF-I and insulin phosporylated their own receptors at low concentrations, 10−10 and 10−9 M, respectively. IGF-I, at low concentrations, 10−9 M, phosphorylated the immunoprecipitate obtained by using insulin receptor antibodies. The anti-phosphotyrosine immunoreactive bands on Western blot were broader after stimulation with IGF-I in comparison with insulin which could indicate a phosphorylation of both IGF-IR and IR tyrosine kinases. These observations are compatible with the presence and activation of hybrid IGF-I/insulin receptors by IGF-I (Moxham et al., 1989; Soos and Siddle, 1989). By immunoprecipitating either the IGF-I receptor or the insulin receptor and blotting the membranes with antibodies against both receptors, we could demonstrate both IGF-I and insulin -subunit receptor protein. This is indicative of the presence of IGF-I/insulin hybrid receptors in human endothelial cells, as shown in other tissues (Bailyes et al., 1987; Pandini et al., 2002). To the best of our knowledge, IGF-I/insulin hybrid receptors have not previously been demonstrated in vascular endothelial cells. The slight difference in the molecular weight of the receptors excludes cross-reactivity of the antibodies in the detection step as an explanation for our findings. Cross-reactivity in the immunoprecipitation step could be alternative explanation. However, the same result was obtained when non-cross-reactive monoclonal antibodies were used (Prigent et al., 1990; Soos et al., 1992). It has been suggested that the formation of IGF-I/insulin hybrid receptors influences the effects of insulin and IGF-I at the tissue level (Federici et al., 1997). A larger fraction of IR has been described to be present as hybrid receptors when the amount of IGF-IR is higher than the amount of IR (Bailyes et al., 1997). The affinity of the IGF-I/insulin hybrid receptors for insulin and IGF-I are reported to be similar to the affinity of IGF-I receptors (Soos et al., 1993; Seely et al., 1995). The IR could be activated by low concentrations of IGF-I 10−9 to 10−8 M whereas the IGF-I receptor was not activated by insulin. This could reflect the transautophosphorylation of the IR -subunit after IGF-I binding to the IGF-IR ␣-subunit in IGF-I/insulin hybrid receptors (Frattali and Pessin, 1993). Our results suggest the presence of functional IGF-I/insulin hybrid receptors, which might diminish the effects of insulin in endothelial cells.
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The gene expression of IGF-IR measured by real-time RT PCR was several folds higher than that of IR in HUVEC cultured for two to three passages. This is in agreement with results in other endothelial cells (Chisalita and Arnqvist, 2004). In freshly isolated HUVEC, the amount of IGF-I receptor mRNA also was several fold higher than insulin receptor mRNA indicating that the predominating gene expression of IGF-IR is also present in vivo and not due to cell culture. The higher specific binding of IGF-IR compared to IR is in agreement with the results of Zeng and Quon (1996). They measured IGF-I and insulin-specific binding in HUVEC and observed that IGF-IR was about 10 times more abundant than IR on HUVEC. The specific binding of IGF-I in HUVEC fitted a one site binding model with an affinity corresponding to the binding of IGF-I to isolated IGF-IR (Kurtzhals et al., 2000). We found that the IGF-I receptor already was phosphorylated by IGF-I at a concentration of 10−10 M IGF-I which suggests that the IGF-I receptor is activated at physiological concentrations of free IGF-I present in vivo (Frystyk et al., 1994). Human insulin interacted with the IGF-IR with over thousand folds less potency than IGF-I itself. The binding of insulin was low but clearly displaced by unlabelled insulin with an IC50 value corresponding to the insulin receptor (Kurtzhals et al., 2000). The immunoprecipitated insulin receptor was found to be phosphorylated by insulin at concentrations of 10−9 to 10−8 M but not consistently with 10−10 M insulin. This indicates activation of the insulin receptor in the high physiological range of circulating insulin (Olsson et al., 1988). These insulin concentrations are lower than those required for cross-activation of the IGF-IR (Chisalita and Arnqvist, 2004), which suggests that some IR homodimers are present and that not all IR are part of IGF-I/insulin hybrid receptor complexes. The abundance of IGF-IR compared to IR, the presence of IGF-I/insulin hybrid receptors that appear to be activated by physiological concentrations of IGF-I but not by insulin, and the effect of low concentrations of IGF-I on endothelial cells suggest that IGF-I is of importance for the regulation of human endothelial cell function. Clinical conditions with low circulating IGF-I levels, such as type 1 diabetes (Juul, 2003), growth hormone deficiency and old age (Khan et al., 2002), are associated with an increased prevalence of cardiovascular diseases. A possible mechanism contributing to cardiovascular disease for this could be an impaired NO-production in endothelial cells as a result of low IGF-I levels (Zeng and Quon, 1996). Studies on the effect of the knock-out of IGF-IR in vascular endothelial cells have been performed in mice (VENIFARKO mice) by Kondo et al. (2004) that also point to an effect of IGF-IR on NO production. The IGF-IR knock-out mice reportedly responded with a smaller increase of eNOS levels in retinal endothelial cells when subjected to relative hypoxic stress as compared to control mice. The role of IGF-IR and IR in vascular endothelial cells in health and disease will have to be the subject of future studies.
Acknowledgements We are grateful to Anna-Kristina Granath for excellent technical assistance. We would like to thank Prof. K Siddle for providing us with the monoclonal antibodies. Financial support was obtained form, the Swedish Medical Research Council (04952), the Swedish Diabetes Association, Barndi¨ abetes Fonden and the County of Osterg¨ otland.
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Soos, M.A., Field, C.E., Lammers, R., Ullrich, A., Zhang, B., Roth, R.A., Andersen, A.S., Kjeldsen, T., Siddle, K., 1992. A panel of monoclonal antibodies for the type I insulin-like growth factor receptor. Epitope mapping, effects on ligand binding and biological activity. J. Biol. Chem. 267, 12955–12963. Soos, M.A., Field, C.E., Siddle, K., 1993. Purified hybrid insulin/insulinlike growth factor-I receptors bind insulin-like growth factor-I, but not insulin, with high affinity. Biochem. J. 290, 419–426. Treadway, J.L., Morrison, B.D., Soos, M.A., Siddle, K., Olefsky, J., Ullrich, A., McClain, D.A., Pessin, J.E., 1991. Transdominant inhibition of tyrosine kinase activity in mutant insulin/insulin-like growth factor I hybrid receptors. PNAS USA 88, 214–218. Zeng, G., Quon, M.J., 1996. Insulin-stimulated production of nitric oxide is inhibited by wortmannin. Direct measurement in vascular endothelial cells. J. Clin. Invest. 98, 894–898.
IGF-I/insulin hybrid receptors in human endothelial cells Marloes Dekker Niterta , Simona I. Chisalitaa , Karolina Olssona , Karin E. Bornfeldtb , Hans J. Arnqvista,∗ a
Diabetes Research Center and Division of Cell Biology, Department of Biomedicine and Surgery, Faculty of Health Sciences, Link¨oping University, S-581 85 Link¨oping, Sweden b Department of Pathology, P.O. Box 357470, University of Washington School of Medicine, Seattle, WA 98195-7470, USA Received 23 June 2004; received in revised form 7 October 2004; accepted 12 October 2004
Abstract Vascular complications are common in diabetes. IGF-I receptors (IGF-IR) and insulin receptors (IR) in endothelial cells might respond to altered levels of IGF-I and insulin, resulting in altered endothelial function in diabetes. We therefore studied IGF-IR and IR gene expression, ligand binding, receptor protein, and phosphorylation in human umbilical vein endothelial cells (HUVEC). IGF-IR mRNA was more abundant than IR mRNA in freshly isolated HUVEC (IGF-IR/IR ratio 7.1 ± 1.5) and in cultured HUVEC (ratio 3.5 ± 0.51). Accordingly, specific binding of 125 I-IGF-I (0.64 ± 0.25%) was higher than that of 125 I-insulin (0.25 ± 0.09%). Protein was detected for both receptors and IGF-I/insulin hybrid receptors. IGF-IR phosphorylation was stimulated by 10−10 to 10−8 M IGF-I. IR were activated by 10−9 to 10−8 M insulin and IGF-I. We conclude that HUVEC express more IGF-IR than IR, and also express hybrid receptors. Both IGF-I and insulin phosphorylate their own receptors but only IGF-I seems to phosphorylate hybrid receptors. © 2004 Elsevier Ireland Ltd. All rights reserved. Keywords: IGF-I receptor; Insulin receptor; Insulin/IGF-I hybrid receptor; Endothelial cells; Human
1. Introduction The endothelium is a dynamic tissue which plays a major role in the regulation of vascular tone; it also has a function in haemostasis, permeability and inflammation. Endothelial dysfunction is considered to play an important role in the development of atherosclerosis (Cines et al., 1998; Hsueh and Quinones, 2003). Hyperinsulinemia, insulin resistance as well as altered IGF-I levels have been implicated in the pathogenesis of cardiovascular disease (Dandona et al., 2003; Juul et al., 2002), although the mechanisms are not clear. Moreover, the presence and function of IGF-I receptors (IGF-IR) and insulin receptors (IR) in endothelial cells are not well characterised. The IGF-IR and IR are homologous tetrameric receptors, with two heterodimers consisting of an extracellular ␣-subunit and a transmembrane -subunit. The ␣-subunit ∗
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0303-7207/$ – see front matter © 2004 Elsevier Ireland Ltd. All rights reserved. doi:10.1016/j.mce.2004.10.003
contains the ligand-binding site; the -subunit has the ligandsensitive tyrosine kinase site that is trans-autophosphorylated after ligand binding (Treadway et al., 1991; Frattali et al., 1992). Both IGF-I and insulin bind to either receptor, but the affinity for their cognate receptor is 100–1000 times higher. Hybrid IGF-I/insulin receptors with one IGF-IR heterodimer and one IR heterodimer have also been found, particularly in tissues where IGF-IR numbers are significantly different from IR numbers (Bailyes et al., 1997; Federici et al., 1997). Hybrid IGF-I/insulin receptors are reported to behave in a manner similar to IGF-IR with respect to ligand-induced autophosphorylation, receptor internalisation, and degradation (Seely et al., 1995). The presence of hybrid IGF-I/insulin receptors and their proportion to IGF-IR and IR could influence the cellular effects of IGF-I and insulin. We investigated the presence of IGF-IR, IR, and hybrid IGF-I/insulin receptors in human umbilical vein endothelial cells (HUVEC). We also studied whether IGF-I and insulin could stimulate tyrosine phosphorylation of the receptors.
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2. Materials and methods
2.3. Real time RT-PCR analysis
2.1. Materials
The freshly isolated or cultured cells were washed with PBS and total RNA was harvested with the Qiagen RNAeasy mini kit (Qiagen GmbH, Hilden, Germany). One micrograms of RNA was reverse transcribed with random primers and Superscript II Rnase H reverse transcriptase. The cDNA was stored in aliquots at −20 ◦ C until use. Comparative quantitative PCR analysis was performed with primer/probe pairs for IGF-I, insulin receptor and IGFI receptor mRNA. The reaction consisted of 25 ng cDNA, 50 nM probe, 300 nM of sense and anti-sense primers, 12.5 l 2× TaqMan mastermix (PE Applied Biosystem, Stockholm, Sweden), and water in a total volume of 25 l. The following primers and probes were used: IGF-I receptor forward primer: 5 -TTT CAA CAA GCC CAC AGG GT-3 , IGF-I receptor reverse primer: 5 -CCA CGA TGC CTG TCT CAC G-3 , IGF-I receptor probe: 5 (FAM)-TGG CTC CAG CAG TCG GAG GGC (TAMRA)-3 ; insulin receptor forward primer: 5 -AGG AGC CCA ATG GTC TGA-3 , insulin receptor reverse primer: 5 -CAG ACG CAG AGA TGC AGC-3 and insulin receptor probe: 5 (FAM)-ACC ATA TCG CCG ATA ACT CAC TTC ATA CAG (TAMRA)-3 . The PCR program started with an initial temperature of 50 ◦ C for 2 min and increased to 95 ◦ C for 10 min, followed by 40 amplification cycles of 15 s at 95 ◦ C and 1 min at 60 ◦ C. An ABI Prism 7700 sequence detector equipped with a 96-well thermal cycler connected to Sequence Detector v1.6.3 software (Applied Biosystems, Stockholm, Sweden) was used in the experiments. The relative IGF-I, insulin receptor and IGF-I receptor mRNA amount was normalised to the housekeeping gene encoding GAPDH. GAPDH reactions of all samples were run in the same 96-well plate in the same run as the reactions of the samples for the genes of interest. 1.25 l GAPDH mastermix (PE Biosystem) consisting of primers and probe for GAPDH was added to 25 ng of cDNA, 12.5 l TaqMan mastermix and water for a total reaction volume of 25 l. Reactions were performed in duplicate. The relative quantity of, insulin receptor and IGF-I receptor mRNA was calculated using the comparative CT method after initial experiments showed similar quantitative PCR efficiency rates for IR, IGF-IR and GAPDH.
Primers and probes were purchased from SGS (Scandinavian Gene Synthesis AB, K¨oping, Sweden). Mono-125 I(Tyr A14)-human insulin (1500 Ci/mmol) and 125 I-IGF-I (2000 Ci/mmol) were obtained from Amersham Pharmacia Biotechnology (Buckinghamshire, UK). The following antibodies were obtained from Santa Cruz biotechnology (Santa Cruz, CA): Polyclonal anti-IGF-IR -subunit antibodies (C20, sc-713) and polyclonal anti-IR -subunit antibodies (C19, sc-711) used for immunoprecipitation and immunoblot. A monoclonal anti-IGF-IR ␣-subunit antibody (2C8, sc-463) and a monoclonal anti-IR -subunit antibody (29B4, sc-09) used for immunoprecipitation, and an anti-phosphotyrosine antibody (PY20, sc-508) used for immunoblot. The monoclonal anti-IR ␣-subunit 83-7 antibody and monoclonal anti-IGF-IR 17-69 antibody were kind gifts of Prof. K. Siddle (Cambridge University, Cambridge, UK). Goat anti-rabbit-HRP (Zymed, San Francisco, CA), sheep anti-mouse HRP (Amersham, Bucks, UK) and streptavidinHRP (Amersham, Bucks, UK) were used as secondary antibodies. Enhance chemiluminescence detection system was obtained from Amersham Pharmacia Biotechnology (Bucks, UK).
2.2. Cell culture EC were isolated from human umbilical veins by a 15 min collagenase digestion (0.5 mg/ml DMEM containing 25 mM HEPES). Briefly, the umbilical veins were collected in a sterile container with PBS and penicillin (100 IU/ml), streptomycin (100 IU/ml) and gentamycin (10 g/ml) at 4 ◦ C. Each umbilical vein was washed with sterile PBS and then treated for 15 min with 100 mg collagenase H in medium A (200 ml DMEM containing 5 ml HEPES and 10 IU/ml penicillin and 10 IU/ml streptomycin) at 37 ◦ C. The cells were collected in 10 ml medium A and the veins were washed with sterile PBS and the flow-through was also collected. The cells were centrifuged at 500 × g for 5 min, washed with culture medium and plated out in culture flasks coated with 0.2% gelatine. Freshly isolated cells were collected for experiments after the second centrifugation. The cells were cultured in DMEM supplemented with 8% heat-inactivated foetal calf serum (FCS) and EGM-2 singlequots® (Bio-Whittaker, Cambrex, Walkersville, MD, USA) with 0.04% hydrocortisone, 0.04% human fibroblast growth factor, 0.1% vascular endothelial growth factor, 0.1% R3 -IGF-I, 0.1% ascorbic acid, 0.1% human epithelial growth factor, 0.1% gentamycinamphotericin, 2 g/ml fungizone, and 0.1% heparin was also added. Cultured cells were used between passages 1 and 2. Cultured control cells were subjected to DMEM medium containing 2% FCS and fungizone for 24 h prior to harvesting of the cells.
2.4. Binding assays HUVEC were cultured to near-confluence in six-well plates and incubated for 4 h at 10 ◦ C in HEPES binding buffer, pH 7.8, of the following composition (mmol/l): HEPES 100, NaCl 120, KCl 5, MgSO4 1.2, glucose 8 and 0.1% bovine serum albumin with the addition of 125 I-IGF-I (12.5 × 10−12 M) or 125 I-insulin (43.9 × 10−12 M) and unlabeled polypeptides at indicated concentrations. The cells were then washed 4 times with ice-cold PBS buffer and dissolved in 0.1% SDS and the radioactivity was measured in a gamma counter. Unspecific binding was defined as binding
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of 125 I-IGF-I or 125 I-insulin in the presence of 10−7 M unlabeled IGF-I or insulin. The binding data were analysed using GraphPad Prism program (GraphPad Software Inc., San Diego, USA) and are expressed either as per cent of the total 125 I-polypeptide bound (in displacement curves) or as percent specific binding of total 125 I-polypeptide added to the medium.
2.5. Immunoprecipitation and Western blot analyses Stimulation of receptor tyrosine kinase phosphorylation was studied in confluent HUVEC that were exposed to lowserum (2%) DMEM for 18 h. The cells were washed with fresh low-serum medium and incubated with 50 M Na3 VO4 in low-serum medium containing 1 mg/ml BSA for 30 min on ice. The cells were then stimulated with insulin or IGF-I in pre-warmed low-serum medium at indicated concentrations or subjected to pre-warmed low-serum medium only for 10 min at 37 ◦ C .1.5 ml lysis buffer (20 mM Tris–HCl, pH 7.5, 150 mM NaCl, 5 mM EDTA, 0.5% sodiumdeoxycholate and 0.5% Triton X-100 supplemented with 1 mM phenylmethylsulfonyl fluoride (PMSF), 1 mg/ml leupeptin, 200 mM Na3 VO4 , 1 mg/ml aprotinin) was added to the cells and incubated for 30 min on ice. Cell lysates were then harvested, centrifuged at 4 ◦ C at 20,000 × g for 15 min and stored at −70 ◦ C until use. The protein content of the samples was determined with the bicinchoninic acid method (Pierce, Rockford, IL, USA). For immunoprecipitation experiments, 0.5 ml cell lysate (containing approximately 500 g of protein) was incubated with 1.25 l immunoprecipitating antibody bound to protein A sepharose for the polyclonal rabbit antibodies or to protein G sepharose for the monoclonal mouse antibodies. The immunoprecipitates were collected, washed and diluted in 25 l of 2× Laemmli sample buffer. The immunoprecipitated samples were boiled for 3 min, centrifuged and separated on a 7.5% SDS–PAGE (Bio-Rad). The samples were electrotransferred onto a PVDF membrane and unspecific antibody binding was blocked by a 60 min-incubation of the membrane with 5% non-fat dry milk in TBS-0.1% Tween 20. The membrane was incubated overnight at 4 ◦ C with primary antibody, followed by a 60 min incubation with secondary antibody. Detection of the protein was performed by enhanced chemiluminescence. Between the steps the membrane was washed with TBS-0.1% Tween 20 wash buffer. The antibodies were diluted in wash buffer. Secondary antibody dilutions contained 2.5% non-fat dry milk except the sheep anti-mouse IgG which was diluted in wash buffer containing 3% bovine serum albumin. Preliminary experiments showed that the supernatant of the cell lysate after immunoprecipitation still contained IGFIR and IR in amounts that could be immunoprecipitated and blotted (results not shown). Even after a third immunoprecipition with antibodies against IGF-IR and/or IR, the receptors could be shown on immunoblot. We have therefore used
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supernatants of cell lysates, that were previously immunoprecipitated with monoclonal IGF-IR 2C8 or monoclonal IR 29B4, for immunoprecipitation with polyclonal IGF-IR C20 and IR C19 in order to show phosphorylation of the tyrosine kinase of the receptors. After successful blotting with the first primary antibody, the membrane was stripped of antibodies by a 30 min incubation in stripping buffer consisting of 10% SDS, 100 mM S-mercaptoethanol, 62.5 mM Tris–HCl (pH 6.7) at 55 ◦ C. In order to detect hybrid receptor, the membrane was subsequentially blotted with primary antibody against the other receptor. 2.6. Statistical analysis Values are given as mean ± S.E.M. Potential differences were analysed with Student’s t-test with P < 0.05 considered significant.
3. Results 3.1. Gene expression Detectable levels of mRNA for IGF-IR and IR were demonstrated in total RNA obtained from HUVEC cultured for two passages (Fig. 1). IGF-IR mRNA was more abundantly expressed than IR mRNA. After standardization of the amount of receptor mRNA to the amount of the housekeeping gene GAPDH mRNA, it was found that cultured HUVEC contained approximately 3.5 ± 0.51 (P < 0.001) times more IGF-IR mRNA than IR mRNA. IGF-IR mRNA and IR mRNA were also detected in total RNA obtained from freshly isolated HUVEC (n = 6). Freshly isolated HUVEC contained more IGF-IR mRNA than IR mRNA; 7.1 ± 1.5 (P < 0.001) fold more.
Fig. 1. Expression of IGF-IR and IR mRNA in cultured HUVEC. Results are presented as relative mean amount of mRNA relative to the mean amount of IR mRNA ± S.E.M. (n = 6 for each).
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Fig. 2. Displacement of 125 I-labelled IGF-I from its receptor by addition of increasing concentrations of unlabeled IGF-I or insulin. Near-confluent cells were incubated for 4 h at 10 ◦ C in Hepes buffer (mean ± S.E.M., n = 4).
3.2. Ligand binding The specific binding of 125 I-IGF-I (0.64 ± 0.25%, mean ± S.E.M.) was higher in HUVEC than the specific binding of 125 I-insulin (0.25 ± 0.092%, mean ± S.E.M.). This indicates the presence of a larger amount of IGF-IR than IR on the plasma membrane of HUVEC. Competition studies with unlabeled polypeptide (n = 4) showed a more potent displacement of 125 I-IGF-I with unlabeled IGF-I than with unlabeled insulin (Fig. 2). The IC50 value, indicating the concentration needed to give halfmaximal displacement, was approximately 200-fold lower for IGF-I (3.6 × 10−10 M, confidence interval 3.2 × 10−11 to 4.04 × 10−9 M) than for insulin (8.25 × 10−8 M, confidence interval 3.28 × 10−9 to 2.08 × 10−7 M). The difference was statistically significant (P < 0.0001). Conversely, 125 I-insulin was more effectively displaced by unlabeled insulin than unlabeled IGF-I (Fig. 3). The IC50 value for 125 Iinsulin displacement by insulin was 2.60 × 10−10 M (confidence interval 4.50 × 10−11 to 1.47 × 10−9 M) and for IGFI was 7.39 × 10−9 M (confidence interval 8.30 × 10−10 to 6.5 × 10−8 M), a 30-fold difference in potency (P < 0.0001). Western blot analysis of receptor protein expression and activation.
Fig. 3. Displacement of 125 I-labelled insulin from its receptor by addition of increasing concentrations of unlabeled insulin or IGF-I. Near-confluent cells were incubated for 4 h at 10 ◦ C in Hepes buffer (mean ± S.E.M., n = 4).
Fig. 4. Tyrosine phosphorylation of IGF-IR -subunit and protein expression of IGF-IR -subunit and IR -subunit after immunoprecipitation with IGF-IR C20 antibody. Cells were stimulated with IGF-I or insulin for 10 min at indicated concentrations. Immunoblot with phosphotyrosine PY20 antibody (upper panel), IGF-IR C20 antibody (middle panel) and IR C19 antibody (lower panel). Results are representative of three experiments. M: molecular weight marker; CTL: control; Ins: insulin; the arrow indicates the molecular weight marker at 97 kDa.
IGF-IR protein could be detected in cell lysates from cultured HUVEC after immunoprecipitation with a polyclonal antibody against the IGF-IR -subunit (C20) followed by immunoblotting with the same antibody (Fig. 4, middle panel). IR protein was also found in HUVEC cell lysate after immunoprecipitation and immunoblotting with a polyclonal antibody against the IR -subunit (C19) (Fig. 5, middle panel). The -subunit of the IGF-IR is slightly larger (97 kDa) than that of the IR (95 kDa) and this difference was reflected in the location of the immunoreactive bands on the blot. These results were confirmed after immunoprecipitation with either a monoclonal anti-IGF-IR (2C8) antibody or a monoclonal anti-IR (29B4) antibody followed by immunoblotting with polyclonal antibodies (results not shown). Stimulation of IGF-IR phosphorylation was studied after immunoprecipitation with anti-IGF-IR (C20) and immunoblot with anti-phosphotyrosine (PY20) antibodies. The IGF-IR tyrosine kinase was activated by IGF-I at concentrations ranging from 10−10 to 10−8 M (Fig. 4 upper panel, results of 10−8 M not shown). The IGF-IR did not show phosphorylation after stimulation with the same concentrations
Fig. 5. Tyrosine phosphorylation of IR -subunit and protein expression of IR -subunit and IGF-IR -subunit after immunoprecipitation with IR C19 antibody. Cells were stimulated with insulin or IGF-I for 10 min at indicated concentrations. Immunoblot with phosphotyrosine PY20 antibody (upper panel), IR C19 antibody (middle panel) and IGF-IR C20 antibody (lower panel). Results are representative of three experiments. M: molecular weight marker; CTL: control; Ins: insulin.
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photyrosine immunoreactive bands of IR after stimulation with IGF-I (Fig. 5 upper panel) could represent phosphorylation of the IR -subunit as well as the IGF-IR -subunit of IGF-I/insulin hybrid receptors.
4. Discussion
Fig. 6. Hybrid receptor protein in HUVEC after immunoprecipitation with monoclonal antibodies. Cells were stimulated with insulin or IGF-I for 10 min at indicated concentrations. (A) Immunoprecipitation with IGF-IR 17-69 antibody and immunoblot with IGF-IR C20 antibody (upper panel) or IR C19 antibody (lower panel). (B) Immunoprecipitation with IR 83-7 antibody and immunoblot with IR C19 antibody (upper panel) or IGF-IR C20 antibody (lower panel). Immunoreactive bands are located at different locations on the blot. Results are representative of three experiments. M: molecular weight marker; CTL: control; Ins: insulin.
of insulin. Stimulation of IR tyrosine kinase phosphorylation was determined after immunoprecipitation with anti-IR (C19) and immunoblot with anti-phosphotyrosine (PY20) antibodies. The IR was phosphorylated after stimulation with 10−9 to 10−8 M insulin but not convincingly activated after stimulation with 10−10 M insulin. IGF-I (10−9 and 10−8 M) was also able to cause a phosphorylation of the IR (Fig. 5, upper panel). The lower part of the phosphotyrosine immunoreactive bands in the IGF-I stimulated cells was located at the same level in the blot as the bands of the cells stimulated with insulin. After stimulation with IGF-I, however, the resulting band was broader and more diffuse in the upper margin. When the lysates were immunoprecipitated with polyclonal IGF-IR -subunit antibodies (C20) and subsequently immunoblotted with polyclonal IR -subunit antibodies (C19), a band could be detected on the blot (Fig. 4, lower panel). This band corresponded to the lower molecular weight band of the -subunit of the IR (95 kDa). Immunoprecipitation with polyclonal IR antibodies (C19) and blotting with the polyclonal IGF-I receptor antibody (C20) revealed the presence of a 97 kDa band which matched the molecular weight of the -subunit of the IGF-IR (Fig. 5, lower panel). Identical results were obtained by immunoprecipitating cell lysates with monoclonal antibodies for IGF-IR (17-69) or IR (837), the subsequent immunoblot with polyclonal antibodies against the -subunit of either receptor (C19 or C20) showed the presence of two distinct bands corresponding to 97 and 95 kDa (Fig. 6). The presence of two distinct immunoreactive bands is an indication for the presence of hybrid IGF-I/insulin receptors on HUVEC plasma membrane. The diffuse phos-
In this study, HUVEC were found to express IGF-IR and IR as assessed with gene expression, ligand binding and Western blot. We found higher expression of IGF-IR than IR. A novel finding is the presence of hybrid IGF-I/insulin receptors in human vascular endothelial cells. Both IGF-I and insulin phosphorylate their own receptors but only IGF-I seems to activate hybrid receptors. IGF-I and insulin phosporylated their own receptors at low concentrations, 10−10 and 10−9 M, respectively. IGF-I, at low concentrations, 10−9 M, phosphorylated the immunoprecipitate obtained by using insulin receptor antibodies. The anti-phosphotyrosine immunoreactive bands on Western blot were broader after stimulation with IGF-I in comparison with insulin which could indicate a phosphorylation of both IGF-IR and IR tyrosine kinases. These observations are compatible with the presence and activation of hybrid IGF-I/insulin receptors by IGF-I (Moxham et al., 1989; Soos and Siddle, 1989). By immunoprecipitating either the IGF-I receptor or the insulin receptor and blotting the membranes with antibodies against both receptors, we could demonstrate both IGF-I and insulin -subunit receptor protein. This is indicative of the presence of IGF-I/insulin hybrid receptors in human endothelial cells, as shown in other tissues (Bailyes et al., 1987; Pandini et al., 2002). To the best of our knowledge, IGF-I/insulin hybrid receptors have not previously been demonstrated in vascular endothelial cells. The slight difference in the molecular weight of the receptors excludes cross-reactivity of the antibodies in the detection step as an explanation for our findings. Cross-reactivity in the immunoprecipitation step could be alternative explanation. However, the same result was obtained when non-cross-reactive monoclonal antibodies were used (Prigent et al., 1990; Soos et al., 1992). It has been suggested that the formation of IGF-I/insulin hybrid receptors influences the effects of insulin and IGF-I at the tissue level (Federici et al., 1997). A larger fraction of IR has been described to be present as hybrid receptors when the amount of IGF-IR is higher than the amount of IR (Bailyes et al., 1997). The affinity of the IGF-I/insulin hybrid receptors for insulin and IGF-I are reported to be similar to the affinity of IGF-I receptors (Soos et al., 1993; Seely et al., 1995). The IR could be activated by low concentrations of IGF-I 10−9 to 10−8 M whereas the IGF-I receptor was not activated by insulin. This could reflect the transautophosphorylation of the IR -subunit after IGF-I binding to the IGF-IR ␣-subunit in IGF-I/insulin hybrid receptors (Frattali and Pessin, 1993). Our results suggest the presence of functional IGF-I/insulin hybrid receptors, which might diminish the effects of insulin in endothelial cells.
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The gene expression of IGF-IR measured by real-time RT PCR was several folds higher than that of IR in HUVEC cultured for two to three passages. This is in agreement with results in other endothelial cells (Chisalita and Arnqvist, 2004). In freshly isolated HUVEC, the amount of IGF-I receptor mRNA also was several fold higher than insulin receptor mRNA indicating that the predominating gene expression of IGF-IR is also present in vivo and not due to cell culture. The higher specific binding of IGF-IR compared to IR is in agreement with the results of Zeng and Quon (1996). They measured IGF-I and insulin-specific binding in HUVEC and observed that IGF-IR was about 10 times more abundant than IR on HUVEC. The specific binding of IGF-I in HUVEC fitted a one site binding model with an affinity corresponding to the binding of IGF-I to isolated IGF-IR (Kurtzhals et al., 2000). We found that the IGF-I receptor already was phosphorylated by IGF-I at a concentration of 10−10 M IGF-I which suggests that the IGF-I receptor is activated at physiological concentrations of free IGF-I present in vivo (Frystyk et al., 1994). Human insulin interacted with the IGF-IR with over thousand folds less potency than IGF-I itself. The binding of insulin was low but clearly displaced by unlabelled insulin with an IC50 value corresponding to the insulin receptor (Kurtzhals et al., 2000). The immunoprecipitated insulin receptor was found to be phosphorylated by insulin at concentrations of 10−9 to 10−8 M but not consistently with 10−10 M insulin. This indicates activation of the insulin receptor in the high physiological range of circulating insulin (Olsson et al., 1988). These insulin concentrations are lower than those required for cross-activation of the IGF-IR (Chisalita and Arnqvist, 2004), which suggests that some IR homodimers are present and that not all IR are part of IGF-I/insulin hybrid receptor complexes. The abundance of IGF-IR compared to IR, the presence of IGF-I/insulin hybrid receptors that appear to be activated by physiological concentrations of IGF-I but not by insulin, and the effect of low concentrations of IGF-I on endothelial cells suggest that IGF-I is of importance for the regulation of human endothelial cell function. Clinical conditions with low circulating IGF-I levels, such as type 1 diabetes (Juul, 2003), growth hormone deficiency and old age (Khan et al., 2002), are associated with an increased prevalence of cardiovascular diseases. A possible mechanism contributing to cardiovascular disease for this could be an impaired NO-production in endothelial cells as a result of low IGF-I levels (Zeng and Quon, 1996). Studies on the effect of the knock-out of IGF-IR in vascular endothelial cells have been performed in mice (VENIFARKO mice) by Kondo et al. (2004) that also point to an effect of IGF-IR on NO production. The IGF-IR knock-out mice reportedly responded with a smaller increase of eNOS levels in retinal endothelial cells when subjected to relative hypoxic stress as compared to control mice. The role of IGF-IR and IR in vascular endothelial cells in health and disease will have to be the subject of future studies.
Acknowledgements We are grateful to Anna-Kristina Granath for excellent technical assistance. We would like to thank Prof. K Siddle for providing us with the monoclonal antibodies. Financial support was obtained form, the Swedish Medical Research Council (04952), the Swedish Diabetes Association, Barndi¨ abetes Fonden and the County of Osterg¨ otland.
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