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The Journal of Immunology, 2000, 164: 113-120.
Copyright © 2000 by The American Association of Immunologists

Developmental Expression of Insulin Receptor Substrate-2 During Dimethylsulfoxide-Induced Differentiation of Human HL-60 Cells1

Daniel H. Schacher*, Roger W. VanHoy*, Qiang Liu*,{ddagger}, Sean Arkins*,§, Robert Dantzer, Gregory G. Freund{dagger} and Keith W. Kelley2,*

* Laboratory of Immunophysiology, Department of Animal Sciences, and {dagger} College of Medicine, Department of Pathology, University of Illinois, Urbana, IL 61801; {ddagger} Department of Hematological Oncology, Cancer Center, Sun Yat-Sen University of Medical Science, Guangzhou, China; § Department of Life Sciences, University of Limerick, Limerick, Ireland; and Institut National de la Recherche Agronomique-Institut National de la Santé et de la Recherche Médicale, Unité de Recherches de Neurobiologie des Comportements, Bordeaux, France


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Insulin receptor substrate-2 (IRS-2) is phosphorylated on tyrosine by a number of cytokine receptors and is implicated in the activation of phosphatidylinositol 3'-kinase (PI3-kinase). Here, we demonstrate that induction of granulocytic differentiation of human promyeloid HL-60 cells leads to an increase in the amount of IRS-2 that is phosphorylated in response to insulin-like growth factor (IGF)-I. Although PI3-kinase is often activated following interaction with IRS-1, we could not detect IRS-1 protein, IRS-1 mRNA, or IRS-1-precipitable PI3-kinase enzymatic activity. However, PI3-kinase activity that was coimmunoprecipitated with either anti-phosphotyrosine or anti-IRS-2 following IGF-I stimulation was increased 100-fold. Heightened tyrosine phosphorylation of IRS-2 during granulocytic differentiation was not caused by an increase in expression of the tyrosine kinase IGF-I receptor, as measured by the amount of both the {alpha}- and ß-subunits. Instead, immunoblotting experiments with an Ab to IRS-2 revealed that induction of granulocytic differentiation caused a large increase in IRS-2, and this occurred in the absence of detectable IRS-1 protein. These IRS-2-positive cells could not differentiate into more mature myeloid cells in serum-free medium unless IGF-I was added. These data are consistent with a model of granulocytic differentiation that requires at least two signals, the first of which leads to an increase in the cytoplasmic pool of IRS-2 protein and a second molecule that acts to tyrosine phosphorylate IRS-2 and enhance granulocytic differentiation.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
A high m.w. cytosolic docking protein, originally designated IL-4 phosphorylated substrate (4PS)3 because it is tyrosine phosphorylated by IL-4 (1), has been purified and cloned from FDC-P2 myeloid progenitor cells (2). Based upon sequence homology to insulin receptor substrate (IRS)-1, 4PS is an isoform of IRS-1 and is now designated IRS-2. Ligands that utilize the common {gamma} ({gamma}c) subunit of the hemopoietin receptor family (including IL-2, IL-4, IL-7, and IL-15) phosphorylate IRS-2 on multiple tyrosine residues following ligand activation of Janus-associated family of protein tyrosine kinases (JAK1) and JAK3 (3, 4, 5). IRS-2 contains 23 potential tyrosine phosphorylation sites and interacts with proteins containing src homology 2 (SH2) and SH3 domains, such as the p85 regulatory subunit of phosphatidylinositol 3'-kinase (PI3-kinase) and the Grb2/Sos complex (6, 7). Indeed, IRS-1 shares with insulin receptors both common (recruitment of PI3-kinase) and distinct (no activation of Stat-1, -2 or -3) signal transduction elements following activation with IFN-{alpha} (8). The amino termini of IRS proteins contain two conserved IRS-homology domains 1 and 2 (IH1 and IH2) resembling pleckstrin homology and phosphotyrosine binding domains (2), respectively. These two domains in IRS-1 promote cell survival (9), and IH1 has been specifically implicated in IRS-2 interactions with tyk-2 (10).

Despite their sequence homology, IRS-1 and IRS-2 may respond differently to TNF-{alpha} stimulation (11). After exposure to TNF-{alpha}, IRS-1-expressing 32D myeloid cells have reduced IRS-1 tyrosine phosphorylation, and IRS-1 acts as an inhibitor of insulin receptor tyrosine kinase activity. In contrast, treatment of cells expressing IRS-2 with TNF-{alpha} does not affect IRS-2 tyrosine phosphorylation or tyrosine kinase activity of the insulin receptor. The emerging view is that the two IRS proteins have differences in function with IRS-1 likely to be more involved in cellular metabolism and growth while IRS-2 may be more important for leukocyte activation and function (12, 13, 14). Although IRS-2 is clearly involved in the activation pathways of a number of ILs, the factors that regulate expression of this protein are as yet unknown. Insulin and insulin growth factor (IGF)-I are the classical ligands for activating IRS-1 and IRS-2 (15, 16, 17). Ligand binding leads to the subsequent transphosphorylation of the two ß-subunits of the IGF-I receptor (IGF-IR) on tyrosine residues 1131, 1135, and 1136 (18, 19, 20, 21). IRS-1 and IRS-2 can then be phosphorylated by the IGF-IR and physically associate with the p85 regulatory subunit of PI3-kinase (17).

IRS-1 tyrosine phosphorylation in response to IGF-I stimulation has been reported to be higher in immature human thymocytes than in peripheral blood T cells (22). These data suggest that the IRS family of cytoplasmic docking molecules may be regulated during development of hemopoietic cells. To address this important possibility in myeloid cells, we used the classic system of differentiating HL-60 promyeloid cells into granulocytes by addition of the polar agent DMSO (23). Since we recently demonstrated that these cells express cell surface IGF-IRs (24), they are likely to provide an excellent tool for determining the regulation of IRS proteins during cellular differentiation. Here, we demonstrate that HL-60 cells do not express IRS-1, but rather utilize IRS-2 to recruit PI3-kinase to phosphorylate phosphatidylinositol within seconds following IGF-I stimulation. Induction of differentiation of these promyeloid cells with DMSO increases the amount of IRS-2 protein that is tyrosine phosphorylated in response to IGF-I. This is caused by an increase in the expression of IRS-2 protein rather than an increase in the amount of IGF-I receptors. These are the first results to establish that expression of an important cytokine signaling molecule, IRS-2, is regulated during hemopoiesis. This regulation is likely to be important functionally since these IRS-2-expressing cells are unable to terminally differentiate into granulocytes unless exogenous IGF-I is added.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Reagents

HL-60, U-937, and U-266 cells were purchased from the American Type Culture Collection (ATCC, Manassas, VA). RPMI 1640 medium was purchased from Media Tech (Herndon, VA) and FBS (<0.06 endotoxin units/ml) from HyClone (Logan, UT). Human holo-transferrin, sodium selenite, sodium bicarbonate, DMSO, penicillin, and streptomycin were purchased from Sigma (St. Louis, MO). Recombinant human IGF-I was obtained from Intergen (Purchase, NY). A variety of Abs were used in flow cytometry, including {alpha}IR3 (mouse IgG1; Oncogene Science, Cambridge, MA) that was used to detect the human IGF-IR, mouse IgG1 (Sigma), rat anti-human CD11b ({alpha} subunit of ß2 integrin CR3 Mac-1, IgG2b; Boehringer Mannheim Biochemicals, Indianapolis, IN), rat IgG2b{kappa}, (PharMingen, San Diego, CA), FITC-conjugated goat F(ab[prime)2 anti-rat Ab for detecting CD11b and FITC-conjugated goat F(ab')2 anti-mouse Ab for detecting the IGF-IR (Cappel, Durham, NC). The anti-phosphotyrosine PY20 mouse IgG2b mAb was obtained from Transduction Laboratories (Lexington, KY), and the rabbit polyclonal IgG Ab to the IGF-IR ß-subunit was obtained from Santa Cruz Biotechnology (Santa Cruz, CA). The rabbit polyclonal IgG anti-IRS-1 carboxyl-terminal and anti-IRS-2 Abs were purchased from Upstate Biotechnology (Lake Placid, NY). Protein concentrations were quantitated using the Micro BCA kit obtained from Pierce (Rockford, IL).

Cellular differentiation and flow cytometry

HL-60, U-937, and U-266 cells were grown in complete medium prepared from powdered RPMI 1640 supplemented with 2.0 g/L of sodium bicarbonate, 100 U/ml penicillin, 100 µg/ml streptomycin, and 10% FBS, which was heat inactivated at 56°C for 30 min. For serum-free (SF) culture, RPMI 1640 was supplemented with 12.5 µg/ml human holo-transferrin and 30 nM sodium selenite. Before induction of cellular differentiation, HL-60 cells were washed 3 times in RPMI 1640 to remove serum factors and were maintained in SF medium overnight. The cells were then induced to differentiate by culturing them for 96 h in the presence of SF medium alone, SF medium with 1.25% DMSO, SF medium with 100 ng/ml (14 nM) IGF-I, SF medium with 1.25% DMSO and 100 ng/ml (14 nM) IGF-I, or SF medium with 1.25% DMSO and 10% FBS. Following differentiation, 2 x 105 cells were washed once with ice-cold PBS (1.5 M NaCl, 19 mM Na2HPO4·H2O, 8.4 mM Na2HPO4 (pH 7.4)) containing 1% FBS (PBS/1% FBS) and resuspended in either 50 µl of the anti-CD11b Ab (4 µg/ml in PBS/1% FBS) or 50 µl of the control IgG2b Ab (4 µg/ml in PBS/1% FBS). The cells were incubated at 4°C for 30 min and then washed 3 times with PBS/1% FBS. After the final wash, the cells were resuspended in 50 µl of a 1:100 dilution of FITC-conjugated goat F(ab')2 anti-rat Ab in PBS/1% FBS and incubated for 30 min at 4°C. Finally, the cells were washed 3 times in PBS/1% FBS and resuspended in 200 µl of fixative (1% paraformaldehyde in PBS) and stored at 4°C until flow cytometric analysis was performed using an EPICS V flow cytometer (Coulter Instruments, Hialeah, FL). Bitmaps were created to include a uniform population of cells similar in size and light scatter. Green fluorescence was monitored by single parameter histogram analysis counting 10,000 cells. Control samples stained with the IgG2b isotype-matched control Ab and the FITC-conjugated secondary Ab were used to determine background fluorescence. Histograms of anti-CD11b and isotype control staining were overlain and analyzed by standard flow cytometric methods, as previously described (24).

Oligonucleotide primers

Oligonucleotide PCR primers were selected with the assistance of the software Primer Designer (Scientific and Educational Software, State Line, PA) and were designed to optimize GC content and melting temperature and minimize hairpin and dimer formation. Primers for human IRS-1 were designed based on the human sequence (25). The 5' primer (5'-AGGAAGAGACTGGCACTGAG-3') begins at nucleotide 3818–3837, and the 3' primer (5'-CGGTTAGGACTGAGGTTCAC-3') initiates at nucleotide 4243–4262. These primers amplify a 445 bp DNA fragment. Two 20-mers (5' primer, 5'-GGAAGGTGAAGGTCGGAGTC-3'; 3' primer, 5'-GCTCCTGGAAGATGGTGATG-3') beginning at positions bp 65 of exon 1 and bp 298 of exon 4, respectively, were synthesized for amplification of a 234-bp fragment of human GAPDH cDNA. All oligonucleotides were synthesized by Operon Technologies (Alameda, CA).

RT-PCR

One microliter of RNA (1 µg) was used for each RT reaction and was treated with 1 unit DNase I for 30 min at 37°C to degrade any residual DNA in the sample. The DNase I was then heat denatured at 95°C, and the RNA sample was placed on ice for addition of the RT reaction mixture. The reaction mixture (20 µl; as previously described in Ref. 26) contained 100 µM dNTPs (United States Biochemical, Cleveland, OH), 1 mM DTT (Life Technologies, Gaithersburg, MD), 100 pM of random hexamers (Pharmacia, Piscataway, NJ), and 200 units of Moloney murine leukemia virus reverse transcriptase in 1x Moloney leukemia virus buffer (Life Technologies). Negative control RT samples included the use of either no RNA or no RT in the reaction mixture. The reaction was performed at 42°C for 1 h and terminated by heating at 95°C for 5 min. PCR amplification was conducted in a 50-µl reaction volume containing 5 µl of reverse transcribed cDNA, 200 µM dNTPs, 1.5 mM MgCl2, 15 pmol of each primer, 1x Taq polymerase buffer, and 1.25 units of Taq DNA polymerase (Life Technologies). The PCR reactions were performed in a PTC-100 Programmable Thermal Controller (MJ Research, Watertown, MA) with the following program: denaturing for 30 s at 95°C, annealing for 30 sec at 58°C, and elongation for 30 sec at 72°C for a total of 30 cycles. PCR products were electrophoresed in 6% polyacrylamide gels, stained with ethidium bromide (0.5 µg/ml), and photographed under UV light.

Immunoprecipitation and immunoblotting

Following washing and incubation in SF medium for 4 h, cells were treated with various amounts of IGF-I for 30 s at 37°C. After stimulation, cells were lysed in a homogenization buffer (10 mM sodium phosphate (Fisher Scientific, Pittsburgh, PA), 1.0% Triton X-100, 0.5% deoxycholic acid, 40 nM leupeptin, 24 trypsin-inhibitory units (TIU) aprotinin, 1 mM PMSF, 1 mM sodium orthovanadate, 10 mM tetrasodium pyrophosphate, and 25 mM benzamidine (pH 7.4) at 4°C (all from Sigma)). Insoluble material was removed by centrifugation, and cell lysates were immunoprecipitated by using Abs directed against phosphotyrosine (PY) (4 µg), IRS-1 (1 µg), or IRS-2 (1 µg) conjugated to a mixture (50 µl per tube) of 1 part of protein-G-Sepharose (Pharmacia Biotech, Piscataway, NJ) and 2 parts of Sepharose CL-4B (Sigma). Following overnight incubation, proteins were washed twice with 100 mM Tris, 0.5 M LiCl2, 0.1% Triton X-100 (pH 7.4) at 4°C and twice with 50 mM Tris and 0.1% Triton X-100 (pH 7.4) at 4°C. After washing, proteins were resuspended in 1x Laemmli dissociation buffer (2.5% SDS, Pierce; 78 mM Tris, 25% glycerol (pH 6.8), Sigma), bromophenol blue (Bio-Rad, Hercules, CA), and 200 mM DTT and boiled for 5 min. Proteins were then separated by SDS-6% PAGE and transferred onto a polyvinyl difluoride (PVDF) membrane (Bio-Rad). Following transfer, membranes were blocked with 0.5% casein (Sigma) in Tris-buffered saline for 3 h at 4°C and then incubated with the immunoblotting Abs (anti-IRS-1; anti-IRS-2; anti-IGF-1Rß; 1 µg/ml) in PBS/0.1% Tween 20/5% nonfat dried milk (Nestlé, Glendale, CA) overnight at 4°C. Proteins were then visualized using enhanced chemiluminescence (ECL) (Amersham International, Buckinghamshire, U.K.).

Measurement of inducible PI3-kinase activity

Washed cells in SF medium were treated with the indicated amount of IGF-I for 30 s and lysed using the homogenization buffer described above plus 1 mM DTT. Following overnight incubation with the appropriate Ab, immunoprecipitated proteins were washed three times with wash buffer A (1% Nonidet P-40 (Sigma), 1 mM DTT, and PBS), three times with wash buffer B (0.5% LiCl2, 1 mM DTT, and 100 mM Tris (pH 7.4)), and three times with wash buffer C (10 mM NaCl, 1 mM DTT, and 10 mM Tris (pH 7.4)). The lipid kinase reaction was initiated by addition of 60 µl of a mixture of 0.33 mg/ml of sonicated L-{alpha}-phosphatidylinositol in kinase buffer (20 mM HEPES, 0.4 mM EGTA, 0.4 mM NaPO4, 10 mM MgCl2, 48 µM [{gamma}-32P]ATP (2.1 µCi/nmol; Amersham) (pH 7.4)). The reactions were conducted at room temperature for 15 min and terminated by the addition of 15 µl of 4 N HCl. Lipids were extracted with 200 µl of a 1:1 mixture of chloroform/methanol, followed by subsequent centrifugation at 14,000 x g for 5 min. The chloroform-containing lipid phase was removed and reextracted using 150 µl of a 1:1 mixture of 1 N HCl/methanol, followed by centrifugation at 14,000 x g for 5 min. Ten microliters of the phosphorylated lipids were resolved using TLC with a chloroform/methanol/ammonium hydroxide (75:58:17) running buffer, as previously described (27, 28). Phosphorylated lipid was detected using Phosphorimager analysis (Molecular Dynamics, Sunnyvale, CA) and autoradiography on XAR film (Eastman Kodak, Rochester, NY).

Statistical Analysis

Data were analyzed using Statistical Analysis Systems software, with Student’s t test used to determine differences between treatments (29).


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
Induction of differentiation in HL-60 cells increases tyrosine phosphorylation of IRS-2 in response to IGF-I

The regulation of IRS-2 expression during cellular differentiation has yet to be reported. We recently demonstrated that HL-60 cells express IGF-I receptors, as determined by flow cytometry (24). We have also observed that the ß-subunit of this receptor becomes tyrosine phosphorylated following IGF-I stimulation (our unpublished data). However, potential downstream signaling involving IRS-1 and IRS-2 docking proteins has not been characterized in HL-60 cells even though differentiation of these cells into granulocytes induced by DMSO greatly increases expression of two unknown high molecular mass tyrosine phosphorylated proteins (165 kDa and 177 kDa; Ref. 30). To determine whether either of these previously reported phosphoproteins might be IRS-1 (predicted molecular mass of 131 kDa and apparent molecular mass of 180 kDa; Ref. 15) or IRS-2 (predicted molecular mass of 145 kDa and apparent molecular mass of 185 kDa; Ref. 2), HL-60 cells were cultured in either SF medium alone or SF medium plus 1.25% DMSO. After 96 h, 3 x 107 cells were washed, incubated in SF medium for 3 h at 37°C, and treated with or without 200 nM IGF-I for 30 sec at 37°C. Cell lysates were then immunoprecipitated using an anti-PY Ab and immunoblotted with an anti-IRS-2 Ab. These experiments revealed that IGF-I alone was capable of minimal but detectable tyrosine phosphorylation of IRS-2 in HL-60 cells (Fig. 1Go) However, following induction of differentiation with DMSO in SF medium, HL-60 cells displayed substantially increased tyrosine phosphorylation of IRS-2 protein as compared with control cells. When cells were differentiated with 1.25% DMSO in 10% FBS, IGF-I caused a similar increase in the tyrosine phosphorylated form of IRS-2 (data not shown). Experiments using a specific anti-IRS-1 Ab for Western blotting yielded no detectable tyrosine phosphorylated IRS-1 (data not shown). Based on these data, we concluded that HL-60 cells must express at least some IRS-2 protein and that the IGF-I-induced tyrosine phosphorylation of IRS-2 is enhanced as HL-60 cells are induced to differentiate with DMSO.



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  		FIGURE 1. Induction of differentiation of HL-60 cells by DMSO increases tyrosine phosphorylation of IRS-2 in response to IGF-I. HL-60 cells were cultured in SF RPMI 1640 medium, SF medium plus 1.25% DMSO, or SF medium plus 1.25% DMSO and 10% FBS for 96 h. Cells (3 x 107) were washed, cultured for 4 h in SF medium, and then incubated in the presence (200 nM) or absence of IGF-I for 30 sec at 37°C. Lysates were immunoprecipitated with an anti-PY Ab, separated by SDS-6% PAGE, and immunoblotted with an IRS-2-specific Ab. Tyrosine phosphorylation of the 185-kDa IRS-2 protein was not detected in the absence of IGF-I treatment, but was readily visible 30 sec after addition of IGF-I. Differentiation of these cells with DMSO increased IRS-2 tyrosine phosphorylation induced by IGF-I compared with control cells.

 
Anti-phosphotyrosine-precipitable PI3-kinase activity is induced by IGF-I in a dose-dependent manner

Since IGF-I stimulated tyrosine phosphorylation of IRS-2 protein, we next investigated whether IGF-I would also induce PI lipid kinase activity by recruitment of PI3-kinase after activation of the IGF-IR. Cells (3 x 107) were incubated with increasing amounts of IGF-I from 0 to 200 nM, and, following lysis, cell lysates were immunoprecipitated with an anti-PY Ab. A lipid phosphorylation assay was then performed on these immunoprecipitates. IGF-I increased PY-precipitable lipid kinase activity in a dose-dependent fashion with a maximal induction of 115-fold ± 16-fold (n = 4; p < 0.01) above basal levels at 200 nM IGF-I (Fig. 2Go). As little as 0.2 nM IGF-I induced detectable activity of PI3-kinase. These data clearly establish the ability of IGF-I to recruit and activate PI3-kinase in these cells.



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  		FIGURE 2. IGF-I induces anti-PY-precipitable PI3-kinase activity in a dose-dependent manner. Following serum-deprivation for 4 h, HL-60 cells were treated with various concentrations of IGF-I (0 to 200 nM) for 30 sec at 37°C. PI3-kinase activity immunoprecipitated with an anti-PY Ab was assessed by measuring phosphorylation of L-{alpha}-phosphatidylinositol (PI). The 32P-labeled PI -phosphate (PIP) was separated by TLC and visualized by autoradiography. The highest concentration of IGF-I increased PI3-kinase activity by 115-fold ± 16-fold (n = 4; p < 0.01), as assessed by Phosphorimager analysis.

 
IRS-2, not IRS-1, activates PI3-kinase activity following treatment with IGF-I

Since IGF-I induced PY-precipitable PI3-kinase activity, we next determined whether the tyrosine phosphorylated protein(s) responsible for this activity was IRS-1, IRS-2, or both. Following stimulation of HL-60 cells with 200 nM IGF-I for 30 sec at 37°C and immunoprecipitation with an Ab specific for the IRS-1 carboxyl terminus (which is not reactive with IRS-2) or normal rabbit serum (NRS), no induction of lipid phosphorylation was evident (Fig. 3GoA; 1.5 ± 0.2 above basal levels; n = 4; p > 0.10). However, this IRS-1 Ab was capable of coimmunoprecipitating PI3-kinase activity in U-266 cells (17-fold ± 5-fold over basal levels; n = 4; p < 0.05), which are known to express IRS-1 (10). We then examined the capability of IRS-2 to recruit PI3-kinase to phosphorylate PI. IGF-I-stimulated cell lysates were immunoprecipitated with NRS or with an Ab specific for IRS-2 (which does not cross-react with IRS-1). Stimulation of HL-60 cells with 200 nM IGF-I induced IRS-2-precipitable kinase activity by 110-fold ± 6-fold (Fig. 3GoB; n = 4; p < 0.01) as compared with unstimulated cells. These experiments establish that IGF-I signaling and the subsequent activation of PI3-kinase activity in HL-60 cells utilizes IRS-2 rather than IRS-1 to activate PI3-kinase.



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  		FIGURE 3. IRS-2, not IRS-1, activates PI3-kinase activity following treatment with IGF-I. HL-60 or U-266 cells were washed, incubated in SF medium for 4 h, and then incubated in the presence or absence of IGF-I (200 nM) for 30 sec at 37°C. A, Cell lysates were immunoprecipitated with an anti-IRS-1 Ab or NRS, and immunoprecipitates were assayed for the ability of the coimmunoprecipitated PI3-kinase to phosphorylate L-{alpha}-phosphatidylinositol. Although anti-IRS-1 immunoprecipitates from IGF-I stimulated U-266 lysates exhibited a 17-fold ± 5-fold (n = 4; p < 0.05) induction in PI3-kinase activity, the anti-IRS-1 immunoprecipitates from IGF-I-stimulated HL-60 cells failed to demonstrate a significant induction in PI3-kinase activity (1.5-fold ± 0.2-fold induction; n = 4; p > 0.10) B, Using anti-IRS-2 to immunoprecipitate whole cell lysates, PI3-kinase activity was measured in IGF-I (200 nM)-treated HL-60 cells. Compared with unstimulated cells, the IRS-2 immunoprecipitates caused a 110-fold ± 6-fold increase in the activity of PI3-kinase (n = 4; p < 0.01).

 
HL-60 cells lack IRS-1 protein and mRNA

Since IRS-1 failed to activate PI3-kinase in HL-60 cells, we determined whether this was due to a failure of IRS-1 to interact with PI3-kinase or simply due to a lack of IRS-1 protein. We performed immunoprecipitation (IP)/immunoblotting on HL-60 cells and in a positive control population of U-266 cells. Cell lysates were immunoprecipitated with anti-IRS-1 Ab or NRS as a negative control (Fig. 4GoA). The immunoprecipitates were then separated by SDS-PAGE and immunoblotted with the anti-IRS-1-specific Ab. Although IP/immunoblotting yielded a protein of ~180 kDa (IRS-1) in U-266 cells, no IRS-1 protein was detected in immunoprecipitates from HL-60 cells.



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  		FIGURE 4. HL-60 cells lack IRS-1 protein and mRNA. A, Whole cell lysates from HL-60 (1 x 108) and U-266 cells (3 x 107) were immunoprecipitated with an anti-IRS-1 Ab or NRS and immunoblotted with the anti-IRS-1 Ab. IRS-1 protein could be detected in only U-266 cells. These data are representative of four independent experiments. B, Total cellular RNA was reverse transcribed, and the resulting cDNA was amplified by 30 cycles of PCR using oligonucleotides specific for IRS-1 or GAPDH. The amplified fragments were analyzed on 6% polyacrylamide gels and stained with ethidium bromide. The 445-bp cDNA fragment for IRS-1 was detected in only U-266 cells. A 234-bp GAPDH fragment was amplified GAPDH from cDNA derived from either HL-60 or U-266 cells. Similar results were obtained in three independent experiments.

 
To further determine whether IRS-1 was expressed in these cells, we performed RT-PCR on total cellular RNA isolated from the same cells. Following reverse transcription, cDNA was amplified using oligonucleotides for IRS-1 or GAPDH. These products were separated in 6% polyacrylamide gels, stained with ethidium bromide, and photographed. Fig. 4GoB shows that a 234-bp GAPDH cDNA fragment was present in both HL-60 and U-266 cells, confirming that the RNA used in these experiments was intact. However, the 445-bp IRS-1 product was present only in U-266 cells. These findings extended the absence of IRS-1 to the mRNA level.

Expression of IGF-I receptors remains unchanged following granulocytic differentiation

We next determined whether the IGF-I-induced increase in tyrosine phosphorylation of IRS-2 caused by DMSO was due to an increase in expression of the tyrosine kinase IGF-IR. Cells were cultured in the presence or absence of 1.25% DMSO for 96 h in SF medium. Using flow cytometry, the percentage of cells positive for the IGF-IR was determined by staining with a mAb specific for the extracellular ligand-binding {alpha}-subunit of the IGF-IR. Background fluorescence, as determined with an isotype-matched control mAb, amounted to 5% of the cells (Fig. 5GoA). The small increase in mean fluorescence intensity that occurred in DMSO-treated cells was the result of greater autofluorescence of differentiating cells rather than an increase in the amount of IGF-I receptor. After correction for this increased autofluorescence with the isotype-control Ab, we found that the proportion of cells expressing the IGF-IR receptor was similar in both control (72%) and DMSO-treated (72%) cells (Fig. 5GoA). These results were confirmed in three independent experiments, in which the proportion of IGF-IR-bearing cells in control and DMSO-treated cells was determined to be 75% ± 5 and 76% ± 9, respectively (p > .10). Cells differentiated in DMSO and 10% FBS also expressed similar percentages of cells positive for the IGF-IR (data not shown). Western blotting of whole cell lysates was then performed using an Ab to the tyrosine-kinase ß-subunit of the IGF-IR. In accordance with the results evaluating expression of the {alpha}-subunit of the receptor, control and DMSO-treated HL-60 cells expressed similar levels of IGF-IR ß-subunit protein (Fig. 5GoB). These experiments established that increased phosphorylation of IRS-2 by IGF-I following the induction of differentiation with DMSO is not a result of increased expression of the tyrosine kinase IGF-IR.



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  		FIGURE 5. Expression of IGF-I receptor {alpha}- and ß-chains is unchanged following induction of differentiation of HL-60 cells. A, Cells were incubated in the presence or absence of DMSO for 96 h in SF medium, stained using an anti-IGF-IR {alpha}-subunit mAb or isotype matched (IgG1) control mAb, and then incubated with an FITC-labeled secondary F(ab')2 Ab fragment. The isotype-matched control IgG1 mAb histogram stained less than 5% of the cells and was designated as background fluorescence. Induction of differentiation with DMSO did not cause a change in expression of the IGF-IR. Similar results were observed in three independent experiments (see Results). B, HL-60 cells incubated for 96 h in control SF medium or DMSO were lysed, and 80 µg of protein was separated using SDS-6% PAGE. After transferring to a PVDF membrane, immunoblotting was performed using an anti-IGF-IR ß-subunit Ab. Incubation with DMSO did not affect expression of the IGF-IR ß-subunit, a result observed in three independent experiments.

 
Induction of differentiation with DMSO increases IRS-2 but not IRS-1 protein expression

Since the increase in tyrosine phosphorylation of IRS-2 following treatment with DMSO was not a result of increased expression of the IGF-IR, we investigated the possibility that DMSO increased the amount of IRS-2 protein. HL-60 cells were cultured in the presence or absence of DMSO for 96 h in SF medium, and 80 µg of protein from whole cell lysates was analyzed by immunoblotting with an anti-IRS-2 Ab (Fig. 6Go). Although IRS-2 protein was detectable in the absence of DMSO, this differentiation stimulus induced a large increase in IRS-2 protein expression as compared with control cell lysates. Similar experiments were performed using the anti-IRS-1 Ab to determine whether IRS-1 protein expression was also induced during the induction of differentiation with DMSO. However, IRS-1 remained undetectable in DMSO-treated cells (Fig. 6GoB), even at twice the amount of protein loaded (data not shown). Also, IRS-1-precipitable PI3-kinase activity did not increase over basal levels following treatment with DMSO (data not shown). These data establish that, although IRS-1 protein cannot be detected, IRS-2 expression increases substantially following induction of differentiation with DMSO.



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  		FIGURE 6. Induction of differentiation increases IRS-2 but not IRS-1 protein expression. A, HL-60 cells were cultured in SF medium in the absence or presence of 1.25% DMSO for 96 h and then lysed. Eighty micrograms of protein from whole cell lysates was separated by SDS-PAGE, transferred to PVDF, and blotted with an anti-IRS-2 Ab. Expression of the 185-kDa IRS-2 protein increased in the presence of DMSO. These results are representative of three independent experiments. B, HL-60 cells were cultured in the presence or absence of DMSO, or DMSO plus 10% FBS, for 96 h and compared with IRS-1-expressing U-266 cells. Equal amounts of protein (80 µg) from whole cell lysates were immunoblotted with an anti-IRS-1 Ab using methods described above. IRS-1 protein was detected in only the positive control U-266 cells.

 
HL-60 cells differentiate into granulocytes only in the presence of both DMSO and IGF-I

A recent report demonstrated that an ectopically expressed IGF-I receptor can induce differentiation of murine 32D cells along the granulocytic pathway in a manner similar to G-CSF (31). Even though the amount of tyrosine kinase receptor remains unchanged in HL-60 cells incubated with DMSO, we speculated that human HL-60 cells might be more responsive to IGF-I as a functional consequence of the increase in IRS-2 protein expression and phosphorylation induced by DMSO. The {alpha} subunit of the ß2 integrin CR3 (CD11b or Mac-1) is absent on immature promyeloid cells and has been widely used to measure the initiation of myeloid differentiation into a more mature phenotype (32, 33). In these experiments, cells were incubated for 96 h in SF medium, SF medium plus DMSO, SF medium plus 14 nM IGF-I, or SF medium plus both DMSO and IGF-I. They were then stained with either an anti-CD11b or an isotype-matched control Ab followed by a secondary FITC-conjugated Ab, and flow cytometric analysis was performed on cells fixed with 1% paraformaldehyde. A representative example of flow cytometric analysis of CD11b expression is shown in Fig. 7Go, and a summary of several independent experiments is presented here. Cells in SF medium alone (10% ± 1; n = 3) or in SF medium plus IGF-I (10% ± 1; n = 3) expressed low levels of CD11b at 96 h. Similarly, cells cultured with only DMSO also expressed low levels of the CD11b marker (8% ± 1; n = 3). However, addition of both IGF-I and DMSO permitted these cells to differentiate into granulocytes, as assessed by a 3.5-fold (35% ± 3; n = 3; p < .05) increase in CD11b above basal levels. These data demonstrate that, although DMSO induces expression of IRS-2, it is insufficient to drive differentiation of these cells along the granulocytic pathway. Addition of IGF-I is needed to permit HL-60 cells to differentiate into granulocytes. This finding suggests that at least two signals are needed for terminal differentiation of granulocytic cells, where the induction of differentiation with DMSO augments expression of the intracellular substrate, IRS-2, which then becomes tyrosine phosphorylated in response to IGF-I and leads to enhanced differentiation of these cells.



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  		FIGURE 7. HL-60 cells fail to differentiate with DMSO in the absence of IGF-I. HL-60 cells (2 x 105 cells/well) were treated with: (A) SF RPMI 1640 medium, (B) 1.25% DMSO, (C) 14 nM IGF-I, or (D) the combination of 14 nM IGF-I plus 1.25% DMSO. Ninety-six hours later, the proportion of cells that differentiate into CD11b-positive cells was determined by flow cytometry. The percentage of cells positive for CD11b was determined by subtracting background fluorescence, as assessed by staining with an isotype-matched control Ab gated at 5%, from the CD11b-stained cells. Both DMSO and IGF-I were required for these cells to differentiate into CD11b-positive cells. These experiments are representative of three identical experiments (results given in text).

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Activation of the IGF-I receptor has very recently been reported to induce differentiation of murine 32D cells along the granulocytic pathway in a manner similar to G-CSF (31). In this murine system, ectopic expression of IRS-1 inhibits granulocytic differentiation following overexpression of the IGF-IR. Although IRS-1 declines following the maturation of thymocytes into peripheral blood T cells (22), nothing is known about the expression of IRS-2 during myelopoiesis. IRS-1 knockout mice do not have major metabolic impairments (34, 35), which suggested there was another high m.w. docking molecule that responds to insulin and IGF-I. IRS-2 has now been shown to substitute for many activities of IRS-1 in IRS-1 knockout mice (6, 7, 34, 35). Furthermore, in addition to IL-4, insulin, and IGF-I, several cytokines are now known to phosphorylate and activate IRS-2, including IL-2, IL-15 (3), and IFN-{alpha} (10). Since IRS-2 was originally identified as a critical signaling molecule activated by the IL-4 receptor in promyeloid cells, we have explored the expression and regulation of IRS-2 as human promyeloid cells differentiate into more mature granulocytes.

To examine the role of IRS-2 during hemopoietic differentiation, we chose the well defined HL-60 human promyeloid cell line, which we have recently shown to express functional receptors for IGF-I (24). Here we measured both the constitutive expression of IRS-1 and IRS-2, as well as the expression of these proteins following the well-characterized DMSO-induced differentiation of these cells into mature granulocytic cells (23). We were surprised to find that DMSO failed to induce differentiation of HL-60 cells into granulocytes when cultured in SF medium (Fig. 7Go). However, expression of IRS-2 increased dramatically after these promyeloid cells were induced to differentiate with DMSO (Fig. 6GoA), and these DMSO-treated cells contained substantially more tyrosine phosphorylated IRS-2 following IGF-I stimulation (Fig. 1Go). These findings are similar to earlier experiments (30) that used {alpha}PY blots to measure expression of the c-fgr protooncogene during granulocytic DMSO-induced differentiation of HL-60 cells. Those authors reported an increase in expression of two high molecular mass phosphoproteins (165 kDa and 177 kDa) following differentiation. Although the nature of these phosphoproteins was unknown, we speculated that these two proteins might be IRS-1 and IRS-2, respectively. The data reported here identify the 177-kDa protein to be IRS-2. Although IGF-I has been shown previously to phosphorylate IRS-2 and to cause it to associate with PI3-kinase (17), IGF-I-phosphorylated IRS-2 has not been shown to directly activate PI3-kinase in these cells. In our experiments, we used an IRS-2 Ab to clearly establish that IGF-I phosphorylates IRS-2 and activates IRS-2 to recruit PI3-kinase, resulting in phosphorylation of phosphatidylinositol. Although the previously reported 165 kDa-phosphoprotein (30) corresponds fairly well to the published migration size of IRS-1, our data show that these cells do not express IRS-1 protein (Fig. 4GoA), mRNA (Fig. 4GoB), or IRS-1-recruited PI3-kinase activity (Fig. 3GoB) nor do they express IRS-1 protein following incubation with DMSO (Fig. 6GoB). It therefore appears that the 165-kDa phosphoprotein is not IRS-1, although we have not ruled out the possibility that this protein might be a previously uncharacterized IRS protein.

The increase in tyrosine phosphorylation of IRS-2 in response to IGF-I following the induction of differentiation with DMSO is not due to an increase in the expression of the tyrosine kinase IGF-IR. Using both flow cytometry to assess the expression of the extracellular {alpha}-chain of the IGF-IR and immunoblotting to measure the intracellular ß-chain, we showed that there are similar levels of IGF-IR protein in both control and DMSO-treated cells. These data support those of Pepe et al. (36), who found that IGF-I binding was not significantly altered by granulocytic differentiation of HL-60 cells. Interestingly, despite the lack of change in IGF-IR expression following treatment with DMSO, IGF-I enhanced the differentiation of these cells. Indeed, we expected to find that DMSO would cause at least modest differentiation of promyeloid HL-60 cells when cultured in SF medium. It is important to note, however, that previous studies used a minimum of 5% FBS to differentiate HL-60 cells, and FBS can contain up to 30 nM IGF-I (37). Although HL-60 cells expressed easily detectable IGF-IR, the cells failed to differentiate in the presence of IGF-I alone. Instead, both DMSO and IGF-I were required to permit granulocytic differentiation, which led to a 3.5-fold increase in CD11b expression compared with cells supplemented with either IGF-I or DMSO alone. Similarly, IGF-I alone for 96 h did not increase IRS-2 expression as compared with unstimulated cells (data not shown), whereas DMSO greatly increased expression of IRS-2 in the absence of IGF-I (Fig. 6GoA). These data establish that neither DMSO nor IGF-I alone can complete terminal granulocytic differentiation. Instead, these results are consistent with the idea that at least two signals are necessary to complete granulocytic differentiation: DMSO acts as the first signal by increasing cytoplasmic IRS-2. The second signal, IGF-I, acts through IRS-2 to enhance maturation of these cells.

HL-60 cells differentiated in the presence of DMSO are well known to display several functional characteristics of granulocytes, including superoxide anion production, phagocytosis, and chemotaxis (38, 39). For example, these cells are 90% positive for superoxide anion production after 96 h in 1.2% DMSO (40). Our data demonstrate that DMSO induces a substantial amount of IRS-2 protein expression and phosphorylation in response to IGF-I stimulation. This increase in IRS-2 expression and IGF-I-induced tyrosine phosphorylation following treatment with DMSO suggests three possible scenarios. First, IRS-2 may be critical for IGF-I-induced differentiation of these cells. Since our data show that addition of IGF-I to cultures with DMSO leads to granulocytic differentiation, whereas IGF-I alone does not, it is likely that the DMSO-induced rise in IRS-2 provides an intracellular substrate to permit IGF-I to cause terminal granulocytic differentiation. Second, IRS-2 may function to enable these cells to respond to the vast array of cytokines that can phosphorylate IRS-2. For example, IFN-{gamma} and growth hormone (GH), both of which prime neutrophils for free radical secretion (41, 42, 43), phosphorylate IRS-1 (44). Miyazaki et al. (30) was able to detect the 177-kDa phosphoprotein as early as 48 h, suggesting that IRS-2 expression is induced early in the differentiation process. Differentiating cells may utilize this IRS-2 protein to respond to other cytokines and differentiation factors as the cell matures. Third, it is likely that DMSO induces the expression of other as yet undefined proteins and that IRS-2 acts in concert with these unknown proteins to lead to the granulocytic differentiation of these promyeloid cells. The data further suggest that cytokines that tyrosine phosphorylate IRS-1 and IRS-2, such as IFN-{alpha} and IFN-{gamma}, might act differently in more differentiated cells that express abundant IRS-2. Indeed, the appearance of specific functions in these differentiated granulocytes, such as superoxide anion production and phagocytosis, suggests that these immunocompetent granulocytic cells might utilize IRS-2 as a downstream docking molecule for several cytokine receptors. This is a strong possibility since IRS-2 has been suggested to be important for immunocompetence in IRS-1 knockout mice.

In summary, these data establish that the expression of IRS-2 is up-regulated during DMSO-induced differentiation of human promyeloid cells. Indeed, since DMSO is unable to differentiate HL-60 cells when cultured in SF medium, induction of IRS-2 is likely to be an important mode of action for this polar differentiation agent. Following the DMSO-induced increase in IRS-2 expression, IGF-I tyrosine phosphorylates IRS-2 and leads to differentiation into more mature CD11b-expressing myeloid cells. All of these events occur in the absence of IRS-1.


   Acknowledgments
 
We acknowledge the instrumentation and expert technical assistance provided by Gary Durack in the University of Illinois Urbana-Champaign Biotechnology Center Flow Cytometry Facility (PHS 1S10 RR02277).


   Footnotes
 
1 This research was supported by grants to K.W.K from the National Institutes of Health (NIH) (AG-06246, DK-49311, and MH-51569) and the Pioneering Research Project in Biotechnology financed by the Japanese Ministry of Agriculture, Forestry and Fisheries and to G.G.F. from NIH (CA 61931). Back

2 Address correspondence and reprint requests to Dr. Keith W. Kelley, University of Illinois, Laboratory of Immunophysiology, 207 Edward R. Madigan Laboratory, 1201 West Gregory Drive, Urbana, IL 61801. E-mail address: Back

3 Abbreviations used in this paper: 4PS, IL-4-phosphorylated substrate; IRS, insulin receptor substrate; PY, phosphotyrosine; IGF-I, insulin-like growth factor-I; PI3-kinase, phosphatidylinositol 3'-kinase; JAK, Janus kinase; SF, serum-free; PVDF, polyvinyl difluoride; PIP, PI3'-phosphate; NRS, normal rabbit serum; IH, IRS-homology; IP, immunoprecipitation. Back

Received for publication June 2, 1999. Accepted for publication August 16, 1999.


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