Archives of Biochemistry and Biophysics 558 (2014) 42–50

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Insulin-like modulation of Akt/FoxO signaling by copper ions is independent of insulin receptor Ingrit Hamann a,1, Kerstin Petroll a,b,1, Larson Grimm a, Andrea Hartwig b, Lars-Oliver Klotz a,c,⇑ a

Faculty of Pharmacy and Pharmaceutical Sciences, University of Alberta, Edmonton, AB, Canada Karlsruhe Institute of Technology, Karlsruhe, Germany c Institute of Nutrition, Department of Nutrigenomics, Friedrich-Schiller Universität Jena, Germany b

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Article history: Received 28 February 2014 and in revised form 19 May 2014 Available online 13 June 2014 Keywords: Insulin signaling PTPase (protein tyrosine phosphatase) ROS Copper ions Hepatoma cells Linsitinib Akt FoxO

a b s t r a c t Copper ions are known to induce insulin-like effects in various cell lines, stimulating the phosphoinositide 30 -kinase (PI3K)/Akt signaling cascade and leading to the phosphorylation of downstream targets, including FoxO transcription factors. The aim of this work was to study the role of insulin- and IGF1-receptors (IR and IGF1R) in insulin-like signaling induced by copper in HepG2 human hepatoma cells. Cells were exposed to Cu(II) at various concentrations for up to 60 min. While Akt and FoxO1a/FoxO3a were strongly phosphorylated in copper- and insulin-treated cells at all time points studied, only faint tyrosine phosphorylation of IR/IGF1R was detected in cells exposed to Cu(II) by either immunoprecipitation/immunoblot or by immunoblotting using phospho-specific antibodies, whereas insulin triggered strong phosphorylation at these sites. Pharmacological inhibition of IR/IGF1R modestly attenuated Cu-induced Akt and FoxO phosphorylation, whereas no attenuation of Cu-induced Akt activation was achieved by siRNA-mediated IR depletion. Cu(II)-induced FoxO1a nuclear exclusion was only slightly impaired by pharmacological inhibition of IR/ IGF1R, whereas insulin-induced effects were blunted. In contrast, genistein, a broad-spectrum tyrosine kinase inhibitor, at concentrations not affecting IR/IGF1R, attenuated Cu(II)-induced Akt phosphorylation, pointing to the requirement of tyrosine kinases other than IR/IGF1R for Cu(II)-induced signaling. Ó 2014 Elsevier Inc. All rights reserved.

Introduction Copper ions may interfere with crucial signaling pathways in mammalian cells, resulting in potentially adverse outcomes such as altered gene expression and proliferation [1]. Exposure to copper ions has previously been demonstrated to modulate stressresponsive pathways, such as mitogen-activated protein kinase pathways, and to affect transcription factors such as AP-1 or NFjB [2–4]. Likewise, insulin signaling in hepatoma cells was shown to be imitated by exposure to copper ions in the absence of insulin: Cu(II) elicited the stimulation of known signaling events downstream of the insulin receptor (IR),2 e.g. the phosphoinositide ⇑ Corresponding author at: Institute of Nutrition, Department of Nutrigenomics, Friedrich-Schiller Universität Jena, Dornburger Str. 29, D-07743 Jena, Germany. Fax: +49 3641 949752. E-mail address: [email protected] (L.-O. Klotz). 1 I.H. and K.P. are equal first authors. 2 Abbreviations used: IR, insulin receptor; PI3K, phosphoinositide 30 -kinase; PTPases, protein tyrosine phosphatases; GSK3, glycogen synthase kinase 3; FoxO, forkhead box, class O; G6Pase, glucose 6-phosphatase; ROS, reactive oxygen species; RTK, receptor tyrosine kinases; IGF1R, insulin-like growth factor-1 receptor; DMEM, Dulbecco’s modified Eagle’s medium; FCS, fetal calf serum; HBSS, Hanks’ balanced salt solution; HRP, horseradish peroxidase; SDS, sodium dodecyl sulfate; BCA, bicinchoninic acid; Ins, insulin; IB, immunoblotting. http://dx.doi.org/10.1016/j.abb.2014.06.004 0003-9861/Ó 2014 Elsevier Inc. All rights reserved.

30 -kinase (PI3K)-dependent phosphorylation and activation of the serine/threonine kinase Akt [5]. Moreover, exposure to Cu(II) caused phosphorylation of glycogen synthase kinase 3 (GSK3) as well as of transcription factors of the forkhead box, class O (FoxO) family [6], both of which are known substrates of Akt. Akt-dependent phosphorylation of FoxO proteins leads to their inactivation and nuclear exclusion [7], which was indeed observed in cells expressing EGFPtagged FoxO1a exposed to Cu(II) [6]. Insulin triggers these same effects, leading to inactivation of FoxOs and attenuation of FoxOdependent expression of genes, such as those of gluconeogenesis enzymes like the catalytic subunit of glucose 6-phosphatase (G6Pase) [8] or of plasma proteins like selenoprotein P [9,10] and the major copper protein in human plasma, ceruloplasmin [11]. It is currently unclear how Cu(II) induces these described insulin-like signaling effects and what the molecular targets of copper ions in cells are that result in the modulation of signaling events. The molecular targets would be of interest for a definition of the mode of action of copper ions. Several reasons point to the insulin receptor as an obvious potential target: (i) copper ions stimulate insulin-like signaling (i.e. activation of PI3K/Akt to a comparable extent, followed by comparable FoxO phosphorylation). (ii) Reactive oxygen species (ROS) and several stressful agents, such as quinones, alkylating

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agents and ultraviolet radiation, have previously been shown to trigger activation of receptor tyrosine kinases (RTK) [12–18]. (iii) Copper ions are redox-active entities potentially triggering ROS formation that could elicit RTK activation [1]. (iv) Insulin receptor is a RTK whose activation may be modulated by ROS, such as hydrogen peroxide [19]. Therefore, we set out to investigate whether copper imitates insulin by acting on the insulin receptor (IR) and the related insulin-like growth factor-1 receptor (IGF1R), thereby causing stimulation of downstream signaling. We here demonstrate that copper ions strongly stimulate insulin-like signaling in a fashion independent of IR and IGF1R.

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FoxO1a-EGFP expression plasmid in serum-free DMEM for 24 h using Nanofectin transfection reagent as described by the manufacturer (PAA). Following transfection, cells were washed with PBS, then incubated in the presence of copper(II) sulfate or insulin for 60 min. Where applicable, cells were incubated in the presence of linsitinib as described above. Fluorescence microscopy of cells expressing EGFP-tagged FoxO1a was performed on an Axiovert Observer.A1 fluorescence microscope (Zeiss, Göttingen, Germany) coupled to an AxioCam MRm camera (Zeiss) using suitable filters. Analysis of EGFP-positive cells was done by counting and separating cells into three categories with respect to the major localization of FoxO1a-EGFP (nuclear, cytosolic or both nuclear/cytosolic). For each determination, approximately 200 cells were counted.

Materials and methods Western blotting Reagents and plasmids All chemicals were from Sigma–Aldrich (Oakville, ON, Canada), if not mentioned otherwise. The insulin receptor tyrosine kinase inhibitor, linsitinib (OSI-906), was from Selleckchem (Burlington, ON, Canada) and the general tyrosine kinase inhibitor, genistein, was from LKT Laboratories (St. Paul, MN, USA). Inhibitors were held as stock solutions in DMSO and diluted into serum-free cell culture media for use. The FoxO1a-EGFP expression plasmid [20] was kindly provided by Dr. Andreas Barthel (Endokrinologikum, Bochum, Germany). Cell culture and fluorescence microscopy analyses HepG2 human hepatoma cells were purchased from the German collection of microorganisms and cell cultures (DSMZ, Braunschweig, Germany) and were held in Dulbecco’s modified Eagle’s medium (DMEM, with 4500 mg/l glucose and 2 mM glutamine, Sigma–Aldrich) supplemented with 10% (v/v) fetal calf serum (FCS) (PAA, Etobicoke, ON, Canada), 1% penicillin/streptomycin (Life Technologies, Burlington, ON, Canada) and 1% non-essential amino acids (Sigma–Aldrich), at 37 °C in a humidified atmosphere with 5% (v/v) CO2. Cell viability was assessed using neutral red uptake. HepG2 cells were grown to 60–70% confluence in 24 well-plates, treated with copper for 1 h, washed with PBS and subsequently held in serum-free medium for another 24 h. Cells were then incubated for 2 h with neutral red solution (Sigma–Aldrich; 4 ml of 3.3 g neutral red/l PBS in 100 ml serum-free DMEM). Cells were washed twice with PBS, followed by extraction of neutral red from viable cells by incubation with an ethanol/water/acetic acid (50:49:1, v/ v/v) solution under gentle shaking at room temperature for 2 h. The dye-containing solution was then centrifuged and the absorbance of the cell-free supernatant was measured at 550 nm (with 405 nm as reference). For treatment of cells with copper ions or other agents, HepG2 cells were grown to near confluence, held in serum-free medium for 24 h, followed by the respective treatment. If indicated, cells were preincubated with an inhibitor (genistein or linsitinib) for 60 min prior to the respective treatment with copper or insulin, which was in the continued presence of the inhibitor. DMSO was used as vehicle control. For exposure to copper ions or insulin, cells were washed once with PBS and incubated for 30–60 min in the presence of various concentrations of Cu(II) sulfate or insulin diluted into Hanks’ balanced salt solution (HBSS, Sigma–Aldrich). For exposure to hydrogen peroxide, cells were washed once with PBS and incubated for 30 min in the presence of various concentrations of H2O2 in HBSS. For analysis of FoxO1a-EGFP subcellular localization by fluorescence microscopy, HepG2 cells were grown to approximately 60% confluence in 9 cm2 cell culture dishes and transfected with 3 lg

For analysis of IR, Akt, FoxO1a, FoxO3a and beta-actin levels or modifications, cells were lysed in 2 SDS–PAGE buffer [125 mM Tris/HCl, 4% (w/v) SDS, 20% (w/v) glycerol, 100 mM dithiothreitol and 0.02% (w/v) bromophenol blue, pH 6.8], followed by brief sonication. Samples were applied to SDS–polyacrylamide gels of 10% (w/v) acrylamide, electrophoretically separated and blotted onto nitrocellulose membranes. Membranes were blocked in 5% non-fat dry milk in Tris–buffered saline containing 0.1% (v/v) Tween-20 (TBST) and probed with primary antibody overnight at 4 °C, followed by washing, incubation with secondary antibody [horseradish peroxidase (HRP)-conjugated anti-rabbit IgG or HRP-coupled anti-mouse IgG, GE-Healthcare (Piscataway, USA)] and detection using chemiluminescent HRP substrate. The following primary antibodies were used: anti-phospho-IR-b/IGF1R-b (Y1150/1151)/(Y1135/1136), anti-total-IR-b, anti-phospho-Akt (Ser473), anti-total-Akt, anti-phospho-FoxO1a/FoxO3a (T24/T32), anti total FoxO1a (all from Cell Signaling Technology, Danvers, MA, USA), anti-b-actin (Sigma–Aldrich) and GAPDH (Millipore, Billerica, MA, USA). All primary antibody incubations were in 5% (w/v) BSA in TBST, and all secondary antibody incubations were in 5% (w/v) non-fat dry milk in TBST. General tyrosine phosphorylation was detected using a mouse monoclonal anti-phosphotyrosine antibody, ‘‘4G10 Platinum’’, which is a mixture of two anti-pY antibody clones, 4G10 and PY20 (Millipore). Test for protein tyrosine phosphatase (PTPase) inhibition Tyrosine phosphorylation of IR/IGF1R was stimulated by incubation of cells with insulin (100 nM) for 30 min. Insulin treatment was in the absence or presence of the known phosphatase inhibitor, vanadate (sodium orthovanadate at a final concentration of 1 mM) or the compound(s) of interest whose PTPase inhibitory activity was being investigated. To prevent any further autophosphorylation, cells were treated with 10 lM linsitinib. After 5 min, medium was quickly removed, cells washed with PBS and lysed in 2 SDS–PAGE sample buffer, followed by detection of phospho-IR-b/IGF1R-b (Y1150/1151)/(Y1135/1136) and b-actin by Western blotting. Immunoprecipitation For immunoprecipitation, cells were grown to 70% confluence in 58 cm2 culture dishes as described above. After treatment, cells were washed once with PBS and lysed in 500 ll RIPA buffer (1% IGEPAL CA-630, 0.5% sodium deoxycholate, 0.1% sodium dodecyl sulfate (SDS), 150 mM NaCl, 50 mM Tris–HCl (pH 8), 5 mM sodium fluoride, 1 mM sodium vanadate, 1 mM b-glycerophosphate, 2.5 mM sodium pyrophosphate, 1 lg/ml aprotinin, 1 mM phenylmethylsulfonyl fluoride, 1 mM EDTA and 1 mM DTT), followed by brief sonication. Insoluble material was removed by centrifugation

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Copper ions were previously demonstrated to strongly stimulate the Ser/Thr kinase Akt in a phosphoinositide 30 -kinase (PI3K)-dependent fashion [5]. In order to investigate whether this effect initiates at the level of the insulin receptor (IR) or the related insulin-like growth factor receptor-1 (IGF1R), we tested for a stimulation of IR and IGF1R tyrosine phosphorylation using phosphospecific antibodies. The efficacy of the copper exposure conditions chosen was previously established by us in the same cell culture model employed here [21]. Copper concentrations chosen were between 3 and 100 lM, and while there was no detectable loss in cell viability even 24 h after a 1 h exposure to 10 lM Cu(II), viabilities were lower with 30 lM and 100 lM (with 60% and 20% viability 24 h after exposure, respectively; data not shown). Using a phospho-specific antibody, we analyzed an IR tyrosine residue cluster required to be phosphorylated for full activation

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100 ll transfection mix were added to each well of a 24 well plate. Transfection mix consisted of serum-free cell culture medium (DMEM as described above), 3 ll Dharmafect transfection reagent 4 (Thermo Fisher Scientific, Rockford, IL, USA) and 50 nM SMARTpool ON-TARGETplus INSR siRNA (set of 4 sequences: GAACAAGGCUCCCGAGAGU, AAACGAGGCCCGAAGAUUU, ACGGAGACCUGAAGAGCUA, GCAGGUCCCUUGGCGAUGU) or 50 nM ONTARGETplus non-targeting siRNA pool (set of 4 sequences: UGGUUUACAUGUCGACUAA, UGGUUUACAUGUUGUGUGA, UGGU UUACAUGUUUUCUGA, UGGUUUACAUGUUUUCCUA) (all from Thermo Fisher Scientific). Trypsinized HepG2 cells were diluted in 400 ll DMEM cell culture medium containing 10% FBS and added to each well, yielding a cell confluence of 25%. 48 h posttransfection, cells were washed with PBS and treated with 10 or 100 lM CuSO4 or 100 nM insulin in HBSS buffer for 60 min. Cells were washed with PBS and lysed in 0.5% SDS. Following protein determination, lysates were mixed with 4 SDS–PAGE sample buffer, followed by detection of total InsR, phospho-IR-b/IGF1R-b (Y1150/1151)/(Y1135/1136), phospho-Akt (Ser473), anti-phosphoFoxO1a/FoxO3a (T24/T32) and GAPDH by Western blotting.

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of the receptor upon stimulation and that contains Tyr-1150 and Tyr-1151 (numbers referring to the short, IR-A isoform, corresponding to tyrosines 1162/1163 in IR-B). These sites correspond to Tyr-1135/Tyr-1136 in IGF1R, whose phosphorylation would be detected by the same antibody. Stimulation of HepG2 cells with insulin induced a strong phosphorylation of these tyrosines, whereas copper elicited a slight increase in tyrosine phosphorylation (between approx. 1.5 and 2.5-fold over control; Fig. 1A). This is in stark contrast, however, to the observed strong Akt phosphorylation at Ser-473 in cells exposed to copper ions, which starts at less than 10 lM and even exceeds the effect elicited by insulin (Fig. 1B). Neither a 5, 30 nor 60 min exposure to copper ions elicited significant IR/IGF1R phosphorylation (i.e. no more than in Fig. 1A), whereas there was (i) a distinct activation of Akt by copper ions and (ii) a strong stimulation of IR phosphorylation by insulin at all times (data not shown). In order to test whether any other insulin receptor tyrosine residues might be phosphorylated upon exposure of cells to copper ions, we performed an immunoprecipitation of the insulin receptor, followed by Western blotting analysis of general tyrosine phosphorylation with an anti-phospho-tyrosine antibody. No copper-induced tyrosine phosphorylation of the insulin receptor was detectable, whereas insulin expectedly caused a significant tyrosine phosphorylation of its receptor (Fig. 2A). In order to further substantiate these data, we also used a polyclonal antibody

phospho-IR/IGF1R (rel. signal intensity)

for 10 min at 14,000g and 4 °C. Protein concentration in supernatants was determined using the bicinchoninic acid (BCA) assay (Thermo Fisher Scientific, Rockford, IL, USA), and equal amounts of protein (between 500 and 900 lg, depending on the experiment) from each lysate were incubated with 2 lg of precipitating antibody overnight at 4 °C, as indicated in the respective figures [rabbit monoclonal anti-IR-b (4B8; Cell Signaling) or rabbit polyclonal anti-IR-b (cat # sc-711; Santa Cruz Biotechnology, Santa Cruz, CA, USA)]. Immune complexes were precipitated with protein G magnetic beads (Life Technologies) or protein A/G agarose beads (Santa Cruz Biotechnology), separated from the lysate and washed three times in RIPA buffer. Magnetic or agarose beads were resuspended in 2 SDS–PAGE buffer, heat-denatured, centrifuged and supernatants separated by SDS–PAGE on a 10% polyacrylamide gel, transferred to nitrocellulose membranes and analyzed for tyrosine phosphorylation using the ‘‘4G10 Platinum’’ monoclonal antiphosphotyrosine antibody. Immunoprecipitation was controlled for by reprobing membranes with anti-IR-b (monoclonal or polyclonal). Membrane blocking was in 5% (w/v) BSA in TBST, all primary antibody incubations were in 1% (w/v) BSA in TBST and all secondary antibody incubations were in TBST.

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Fig. 1. Exposure of hepatoma cells to copper: effect on insulin/IGF1 receptor and Akt activation. HepG2 human hepatoma cells were grown to near confluence, washed with PBS and exposed to copper sulfate (3–100 lM in HBSS) for 60 min. After incubation, cells were washed, lysed and analyzed for tyrosine phosphorylation of insulin receptor (IR) and/or insulin-like growth factor-1 (IGF1) receptor and for phosphorylation of Akt by Western blotting and immunodetection employing phospho-specific antibodies. Insulin (100 nM in HBSS, 60 min) served as a positive control. (A) IR phosphorylation at Tyr-1150/1151 and IGF1 receptor phosphorylation at Tyr-1135/1136, as well as total levels of IR and beta actin (as a loading control, not shown) were detected by immunoblotting and signal intensities assessed densitometrically. Phosphorylated receptor/total IR ratios were related to those of the respective control treatments (Ctrl) which were set equal to 1. (B) Phosphorylation of Akt at Ser-473, total levels of Akt and beta actin (as a loading control, not shown) were assessed densitometrically and phospho-Akt/total Akt ratios were related to those of the respective control treatments (Ctrl) which had been set equal to 1. (B, inset) logarithmic presentation of same data. The data shown are means of three independent experiments (±SEM).

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Fig. 2. Effect of copper on insulin receptor tyrosine phosphorylation in HepG2 cells. HepG2 human hepatoma cells were grown to near confluence, washed with PBS and exposed to copper (10, 30, 100 lM in HBSS) for 60 min. Insulin (100 nM in HBSS) served as a positive control. After incubation, cells were washed with PBS, solubilized in RIPA buffer and subjected to immunoprecipitation (IP) followed by immunoblotting. All blots are representative of at least three independent experiments. (A) IR-b (4B8) (monoclonal) immunoprecipitates were further analyzed by Western blotting using anti-phosphotyrosine and anti-IR-b (monoclonal) antibodies. (B) IR-b (polyclonal) (C19) immunoprecipitates were further analyzed by Western blotting using anti-phosphotyrosine and anti-IR-b (polyclonal) antibodies. Tyrosine phosphorylation of IR-b and total levels of IR-b (as loading control) were assessed densitometrically and the phospho-IR b/total IR ratios were related to those of the respective control treatments (Ctrl) which was set equal to 1. The graph shows means of three independent experiments (±SEM).

for precipitation of IR, followed by phospho-tyrosine detection – with a slightly different result: a trend toward tyrosine phosphorylation of the IR was observed in cells exposed to copper ions. Insulin-induced tyrosine phosphorylation of its receptor again was far more substantial than the effect elicited by copper ions (Fig. 2B).

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Fig. 3. Effect of IR inhibitor on IR and Akt phosphorylation in cells exposed to copper. HepG2 human hepatoma cells were grown to near confluence, washed with PBS and exposed to copper (10 and 100 lM in HBSS) or insulin (100 nM in HBSS) for 60 min. Where indicated, the insulin receptor tyrosine kinase inhibitor, linsitinib (OSI-906), was added to culture media to a final concentration of 1 lM 60 min prior to insulin or copper treatment and was further present throughout treatment. DMSO was used as vehicle control (‘‘’’). After incubation, cells were washed, lysed and analyzed for phosphorylation and level of IR (A) and Akt (B) by Western blotting and immunodetection employing phospho-specific antibodies as described in the legend to Fig. 1. All phospho-protein/total protein ratios were related to those of the respective control/DMSO treatments (Ctrl/) which was set equal to 1. (B, inset) Presentation of copper data (10 and 100 lM) from four individual experiments to illustrate inhibitor effect at lower copper concentrations. Copper-induced Akt phosphorylation in the absence of inhibitor was set equal to 1 in each experiment. Data shown in the bar graphs result from densitometric analyses of four independent experiments (±SEM).

Copper-induced signaling: role of insulin receptor Although copper only modestly elevated tyrosine phosphorylation (and activity) of the IR, it cannot be excluded that this minor increase in IR activity is required and sufficient for initiation of Cu signaling. We therefore employed linsitinib (OSI-906), a dual IR/IGF1R inhibitor [22], to test for the role of IR/IGF1R in Cuinduced insulin-like signaling. The concentration of linsitinib that was used (1 lM) was sufficient to fully abrogate both basal and insulin-induced tyrosine phosphorylation of the IR/IGF1R (Fig. 3A). Likewise, insulin-induced Akt activation was blunted in the presence of linsitinib, whereas copper-induced Akt activation was only partly attenuated (Fig. 3B). The above-mentioned slight stimulation of IR/IGF1R tyrosine phosphorylation is again seen in Fig. 3A. Interestingly, whereas the effect on Akt of 100 lM Cu2+ was not significantly blocked by linsitinib, a clear inhibitory effect of the compound on copper-induced Akt activation was detected at 10 lM of Cu2+ in 2 of 4 independent experiments (Fig. 3B, inset). Akt phosphorylates and inactivates transcription factors of the FoxO family, which were previously shown to be phosphorylated and inactivated in cells exposed to copper ions [1,6]. Fig. 4A demonstrates that copper ions induce a strong phosphorylation of FoxO1a and FoxO3a at a site known to be phosphorylated by Akt (Thr-24/Thr-32). At 10 lM Cu2+, this phosphorylation is as strong as the one induced by insulin. Inhibition of IR/IGF1R activity using

linsitinib did not impair copper-induced phosphorylation at 100 lM Cu2+, and no more than an inhibitory trend was observed at 10 lM Cu2+; in contrast, insulin-induced FoxO phosphorylation was back to control levels in the presence of the inhibitor (Fig. 4B). In order to test whether this finding regarding the role of the insulin receptor in FoxO phosphorylation also translates to functional consequences, we tested for alterations in FoxO1a subcellular localization. HepG2 human hepatoma cells were transiently transfected with a plasmid coding for an EGFP-tagged version of FoxO1a. Transfected cells were pretreated with linsitinib or vehicle control (DMSO), followed by addition of insulin/copper in the continued presence of the inhibitor/DMSO. Subcellular localization of FoxO1a-EGFP was then analyzed microscopically. Under basal conditions, FoxO1a-EGFP was predominantly nuclear in roughly 10–15% of all cells analyzed, whereas less than 10% had the protein exclusively cytosolic. Approximately 80% of the cells had both nuclear and cytoplasmic FoxO1a-EGFP. The numbers of cells with nuclear and cytosolic FoxO1a-EGFP were set equal to 1 for control conditions and changes detected upon exposure to copper or insulin were related to these numbers. As expected, and in line with causing Akt-dependent FoxO phosphorylation, insulin stimulated nuclear exclusion of FoxO1a proteins, resulting in a decrease in relative numbers of cells carrying FoxO1a-EGFP predominantly in the nucleus (black bars) and an

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Fig. 4. Effect of copper on Forkhead box, class O (FoxO) phosphorylation – effect of linsitinib. (A) HepG2 cells were grown to near confluence, washed with PBS and exposed to copper (3–100 lM in HBSS) for 60 min. After incubation, cells were washed, lysed and analyzed for phosphorylation of FoxO proteins by Western blotting and immunodetection employing phospho-specific antibodies. Insulin (100 nM in HBSS) served as the positive control. Phosphorylation at Thr 24 (FoxO1a) or Thr 32 (FoxO3a), total levels of FoxO1a and of beta-actin (as a loading control, not shown) were assessed densitometrically and phospho-FoxO1a and 3a/total FoxO1a ratios were related to those of the respective control treatments (Ctrl) which was set equal to 1. The graph shows means of three independent experiments (±SEM). (B) HepG2 cells grown to near confluence were washed with PBS and exposed to copper (10 and 100 lM in HBSS) for 60 min. Insulin (100 nM in HBSS) served as a positive control. Where indicated, the insulin receptor tyrosine kinase inhibitor, linsitinib (OSI-906), was added to culture media to a final concentration of 1 lM 60 min prior to insulin or copper treatment and was present throughout. DMSO was used as vehicle control (‘‘’’). After incubation, cells were washed, lysed and analyzed for phosphorylation and level of FoxO1a/3a employing phospho-specific antibodies as described in (A). Data shown in the bar graphs are means of four independent experiments (±SEM).

increase in numbers of cells with cytosolic FoxO1a-EGFP (gray bars; Fig. 5). Cu at 100 lM even more potently induced FoxO1aEGFP nuclear exclusion, thus imitating insulin. This effect was much less intense at 10 lM Cu, but a trend to nuclear exclusion was still observed. While insulin-induced nuclear exclusion was largely prevented in the presence of linsitinib, there was no effect of the inhibitor on Cu (100 lM)-induced nuclear exclusion, implying that this copper effect is independent of IR/IGF1R. The inhibitor effect on Cu (10 lM)-induced nuclear exclusion was minor (Fig. 5). In addition to using the selective IR/IGF1R inhibitor linsitinib to analyze whether IR phosphorylation and activity are involved in Cu signaling, we used siRNA to knock down endogenous IR. HepG2 cells were transiently transfected with a mixture of four different siRNAs targeting the IR, achieving a downregulation of IR protein levels of about 50–60% (see Fig. 6A and C, detection of IR). As shown in Fig. 6, insulin induced strong phosphorylation of IR/IGF1R (Fig. 6A), of Akt and of FoxO (Fig. 6B) in cells transfected with control (i.e., non-depleting) siRNA, whereas IR-specific siRNA efficiently attenuated insulin-induced phosphorylation of these proteins (see Fig. 6C, white vs. black bars). In sharp contrast, IR knockdown in HepG2 cells did not affect Cu-induced Akt and FoxO phosphorylation, neither at 10 nor 100 lM (Fig. 6B and C, gray vs. black bars).

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Fig. 5. Copper-induced modulation of FoxO1a-EGFP subcellular localization: effect of linsitinib. HepG2 human hepatoma cells were transfected with a FoxO1a-EGFP expression plasmid. After 24 h, insulin (100 nM in HBSS) or copper (10 or 100 lM in HBSS) were added to the cultures for 60 min, followed by fluorescence microscopy. Where indicated, the insulin receptor tyrosine kinase inhibitor, linsitinib (OSI-906), was added to culture media to a final concentration of 1 lM 60 min prior to insulin or copper treatment and was present throughout. DMSO was used as a vehicle control. Approx. 200 EGFP-positive cells were counted for cell classification with respect to predominant localization of EGFP. Data are means of three independent experiments (±SEM).

In summary, copper ions only very modestly stimulate IR/IGF1R phosphorylation in HepG2 cells, and experiments using a pharmacological inhibitor or an siRNA-based approach suggest that the strong stimulation of Akt/FoxO signaling by Cu2+ is largely independent of IR/IGF1R stimulation. The inhibitory action of linsitinib was seen exclusively at lower, non-cytotoxic copper concentrations, suggesting that there is only a minor contribution of IR/IGF1R to Cu-induced Akt signaling, and that with increasing Cu concentrations this contribution is overruled by some copper effect that is yet to be defined. In support of this hypothesis, there was no concentration-dependent increase in the slight Cu-induced IR phosphorylation observed (Figs. 1A, 2B and 3A), whereas such concentration-dependent increase in phosphorylation was obvious for Akt (Fig. 1B) and FoxOs (Fig. 4A). Hence, increasing Cu concentrations would stimulate an effect other than IR/IGF1R activation, resulting in Akt signaling on top of the effects on Akt caused by IR/IGF1R. The concept of a minor or basal activity of IR/IGF1R being required for one layer of Akt signaling whereas contributors other than IR/IGF1R cause the observed strong activation of Akt is in line with our findings in the siRNA experiments. Even after lowering IR levels using siRNA, residual IR levels remained – likely sufficient for basal low IR activity, which might suffice for Cu signaling: different from copper signaling, insulin signaling is strongly dependent on the presence of IR, resulting in the observed attenuation of insulin effects on Akt (Fig. 6). In order to test the hypothesis that copper ions initiate signaling that also causes Akt activation independent of IR/IGF1R on top of the minor contributions of IR/IGF1R, we then tested whether

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Fig. 6. Effect of IR silencing on IR, Akt and FoxO phosphorylation by copper ions. HepG2 cells were transfected with non-depleting control siRNA or IR-specific siRNA for 48 h, washed with PBS and exposed to copper (10, 100 lM) or 100 nM insulin (Ins) for 60 min in HBSS. Cells were washed in PBS and lysed in 0.5% SDS. After protein determination lysates were mixed with 4 SDS–PAGE sample buffer, followed by Western blotting with detection of the amount of (A) total IR protein and InsR/IGF1 receptor, and (B) Akt and FoxO phosphorylation, as well as of GAPDH (loading control). Blots shown are representative of three independent experiments with similar results. (C) Quantitation of signal intensities; signals observed in samples from cells transfected with IR-specific siRNA were related to control siRNA data, which were set to 1. Data shown in the bar graphs result from densitometric analyses of three independent experiments (±SD).

tyrosine kinases other than the IR/IGF1R contribute to Cudependent Akt activation. Copper-induced signaling: role of other tyrosine kinases? As shown in Fig. 7A, an increased extent of general tyrosine phosphorylation of numerous proteins across a broad range of molecular masses is detectable in lysates of cells exposed to Cu(II), implying either a copper-induced tyrosine kinase activation or tyrosine phosphatase inactivation, resulting in a net increase in tyrosine phosphorylation. In order to test whether the stimulation of tyrosine kinases other than IR/IGF1R may be instrumental in Akt activation by copper at all, we used genistein, a general tyrosine kinase inhibitor [23] that, while affecting some insulin-induced biochemical processes, does not appear to potently block autophosphorylation of the insulin receptor itself [24]. At 40 lM, a concentration sufficient to block epidermal growth factor-induced Akt activation in HepG2 cells (data not shown and [25]), insulininduced IR/IGF1R (Fig. 7B), Akt (Fig. 7C) or FoxO (Fig. 7D) phosphorylation were not affected. Despite insulin effects not being altered at this concentration, genistein attenuated Cu(II) (10 and 100 lM)-induced Akt and FoxO (at 10 lM Cu2+) phosphorylation (Fig. 7C/inset and D), implying that tyrosine kinase activity other than the intrinsic activity of the IR or IGF1R contribute to copper-induced activation of Akt. In summary, Cu(II)-induced Akt activation is largely independent of IR/IGF1R, and occurs, at least in part, through protein tyrosine kinases yet to be identified. We then asked how copper can stimulate tyrosine phosphorylation and tested for potential molecular mechanisms. Copper-induced insulin signaling: inhibition of PTPases? Metal ions, including copper ions, can affect cellular signaling cascades in at least two different ways – by stimulating the production of reactive oxygen species (ROS) or by directly binding to proteins involved in signaling (for review, see [26]). One group of proteins that would be affected by both options are protein

tyrosine phosphatases (PTPases). PTPases catalyze the dephosphorylation of phosphotyrosyl residues of protein substrates and are known to be prone to inactivation both by ROS (such as hydrogen peroxide) or by interaction with copper ions, owing to an active site cysteine (see [1,27,28] for review). PTPase inactivation could therefore result in a net enhancement of tyrosine phosphorylation. We set out to test for a contribution of hydrogen peroxide and of PTPases in our setting of copper-induced insulin-like signaling. In order to test whether the formation of hydrogen peroxide in cells exposed to copper ions might be a likely mediator of the observed signaling effects, we exposed cells to hydrogen peroxide. This approach is valid only under the assumption that hydrogen peroxide applied exogenously will reach the cell’s interior. Indeed, hydrogen peroxide permeation of the plasma membrane was found to be ‘‘limited’’ (see [29] for review) – but it is still significant and rapid, especially if facilitated by aquaporins such as AQP3 [30,31] and AQP8 [31,32], both of which are expressed in HepG2 cells [33,34]. Interestingly, phosphorylation of Akt and FoxO, but not the IR/IGF1R was detected in these cells (Fig. 8). However, the concentrations of peroxide required to elicit Akt or FoxO phosphorylation in an extent comparable to that elicited by 10 lM Cu(II) is in the 10 mM range. Even assuming a steep H2O2 concentration gradient across the HepG2 plasma membrane, leading to an assumed intracellular H2O2 steady-state concentration in the high lM region, this would imply that these high lM concentrations would have to be generated in cells exposed to 10 lM Cu(II) (Fig. 8). A similar finding was reported for the epidermal growth factor receptor whose activation by hydrogen peroxide in fibroblasts required high millimolar H2O2 concentrations unless a sensitizing PTPase inhibitor, orthovanadate, was co-applied [17]. We therefore suggest that copperinduced ROS formation is not required for the induction of Akt and FoxO phosphorylation by Cu(II), which is in line with previous findings in HeLa cells and fibroblasts that demonstrated the formation of ROS in cells exposed to copper ions and that this ROS formation observed slower kinetics than the phosphorylation of Akt [5,35].

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Fig. 7. Effect of genistein on IR and Akt activation by copper ions. (A) HepG2 cells were grown to near confluence, washed with PBS and exposed to copper (10, 30, 100 lM in HBSS) for 60 min. After incubation, cells were washed with PBS, solubilized in RIPA buffer and analyzed by immunoblotting (IB) using an anti-phosphotyrosine antibody. The blot shown is representative of three independent experiments. (B–D) HepG2 cells were grown to near confluence, washed with PBS and exposed to copper (10 and 100 lM in HBSS) for 60 min. Insulin (100 nM in HBSS) served as positive control. Where indicated, the general tyrosine kinase inhibitor, genistein, was added to culture media to a final concentration of 40 lM 60 min prior to insulin or copper treatment and was present throughout. DMSO was used as a vehicle control (‘‘’’). After incubation, cells were washed, lysed and analyzed for phosphorylation and level of IR/IGF1R (B), Akt (C) and FoxO1a/3a (D) by Western blotting and immunodetection employing phospho-specific antibodies as described in the legend to Fig. 1. Phospho-protein/total protein ratios were related to those of the respective control/DMSO treatments (Ctrl/) which were set equal to 1. Data shown in the bar graphs result from densitometric analyses of three independent experiments (±SEM).

We then devised an assay (Fig. 9A) to detect whether copper ions interfere with regulatory circuits controlling IR/IGF1R phosphorylation levels at all, i.e. whether tyrosine kinase activity of the IR/IGF1R or PTPases that regulate IR/IGF1R phosphorylation are in any way affected by copper ions. To that end, IR/IGF1R tyrosine phosphorylation was stimulated by the addition of insulin. We then blocked IR/IGF1R tyrosine kinase activity using linsitinib, and followed dephosphorylation of the receptor over time. After 5 min, dephosphorylation almost back to control conditions was achieved (Fig. 9B, lane 3 versus lane 1), suggesting the presence of a PTPase activity that controlled IR/IGF1R tyrosine phosphorylation. The presence of a PTPase inhibitor should attenuate or even block dephosphorylation of IR/IGF1R. We chose orthovanadate, a well-known PTPase inhibitor, as positive control for the assay. As detected in lane 12 of Fig. 9B, tyrosine phosphorylation of IR/IGF1R was indeed upheld if insulin treatment was in the presence of vanadate, even if IR/IGF1R tyrosine kinase activity was blocked by linsitinib. In the presence of copper ions, a similar attenuation of dephosphorylation was observed (Fig. 9B, lanes 6 and 9). Moreover, copper and vanadate ions seem to slightly enhance insulin-induced IR/IGF1R phosphorylation (Fig. 9, lanes 5, 8 and 11; compare with lane 2 for control).

We also tested for Akt phosphorylation (at Ser-473) under the same experimental conditions (Fig. 9B, row 2). As with IR/ IGF1R tyrosine phosphorylation, Akt serine phosphorylation induced by insulin exposure was almost entirely back to control using linsitinib (lane3). Similarly, Akt phosphorylation was maintained in the presence of vanadate – due to some tyrosine phosphatase upstream of Akt that was blocked by vanadate (lane 12). Regarding Cu(II), however, an interference with Akt dephosphorylation cannot be concluded from this experiment since serine (rather than tyrosine) phosphorylation is the crucial activating posttranslational modification of Akt and because, in contrast to vanadate, copper ions strongly induced Akt phosphorylation per se (Fig. 9B, lanes 4 and 7; compare with lane 10 for vanadate). In summary, copper ions significantly impaired IR/IGF1R dephosphorylation in HepG2 cells. Although this cannot fully explain Cu(II)-induced Akt signaling, simply because there is only limited contribution of IR/IGF1R to it, the observed impairment of dephosphorylation provides a molecular mechanism for the minor contribution of IR/IGFR1 to Cu(II)-induced Akt signaling. As all known protein tyrosine phosphatases (PTPases) harbor very similar active sites (see also below), these data also imply that copper ions may impair PTPase activity in general.

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Fig. 9. Analysis of PTPase inhibition by copper ions. (A) Schematic representation of the protein-tyrosine phosphatase (PTPase) inhibition assay. Insulin-induced autophosphorylation of insulin-receptor (IR) is blocked by linsitinib which prevents further phosphorylation. PTPase(s) deactivate(s) IR by dephosphorylating its tyrosines. The potent PTPase inhibitor vanadate inactivates PTPase(s) and thereby prevents dephosphorylation and inactivation of IR. (B) HepG2 cells were stimulated with insulin (100 nM) in the absence or presence of copper(II) sulfate (10 or 100 lM) or sodium orthovanadate (1 mM) for 30 min in HBSS. Linsitinib (10 lM) or DMSO as vehicle control were added. After 5 min, medium was quickly removed; cells were washed with PBS and lysed in 2 SDS–PAGE sample buffer, followed by Western blotting with detection of IR and IGF1 receptor phosphorylation at Tyr1150/1151 and Tyr-1135/1136, respectively, as well as of phospho-Akt at Ser-473 and GAPDH. Blots shown are representative of three independent experiments with similar results.

Copper ion-induced stimulation of insulin-like signaling in human hepatoma cells is demonstrated here to be initiated in a largely insulin/IGF1 receptor-independent manner. Even at concentrations eliciting very strong stimulation of Akt, copper ions cause only a minimal increase in IR tyrosine phosphorylation. Further, complete abrogation of IR or IGF1R activation moderately, but not fully attenuates copper-induced Akt and FoxO phosphorylation. Copper ions, therefore, imitate signaling effects of insulin, but they do not mimic insulin’s mode of action, i.e. they do not fully stimulate its receptor. Although inducing no more than a modest IR/IGF1R tyrosine phosphorylation, copper ions are capable of stimulating significant general tyrosine phosphorylation in cells. The fact that genistein attenuates Cu(II)-induced Akt activation points to a potential role of tyrosine kinases other than the IR/ IGF1R in copper-induced Akt activation. Although copper ions have recently been shown to bind to and stimulate a tyrosine kinase, the dual-specificity (i.e., a tyrosineand Ser/Thr) kinase MEK-1 [36], we are not aware of other examples of copper binding stimulating (rather than inhibiting) a tyrosine kinase. We therefore hypothesize that the effect of copper on overall tyrosine phosphorylation and, more specifically, on tyrosine phosphorylation involved in Akt/FoxO signaling, is due to inactivation of (a) PTPase(s). It has been demonstrated previously that copper ions bind to, and inactivate PTPases, such as PTP1B and human vaccinia H1-related phosphatase (VHR) [1,37]. Most importantly, however, such an inhibition of a PTPase might explain the broad spectrum of proteins whose tyrosine phosphorylation appears to be stimulated in the presence of copper (Fig. 7A): PTPases, while all sharing the active site cysteine thiolate that would allow for an inhibition by copper ions, tend to have multiple substrates. PTPase inhibition would therefore shift tyrosine phosphorylation/dephosphorylation equilibria to the dephosphorylation

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side for multiple tyrosine kinase/substrate pairs. Obviously, the induction of phosphorylation by inhibition of dephosphorylation requires at least basal tyrosine kinase activity – which could explain the modest activation of the IR/IGF1R by copper and the inhibitory effect of genistein on Cu signaling. Regarding the identity of such PTPases, we have observed a distinct inhibitory effect of copper ions on PTPases directly regulating the IR/IGF1R in HepG2 cells (Fig. 9). As copper-induced modulation of Akt is independent of IR/IGF1R, it is unlikely that a PTPase regulating these receptors is the major target causing Akt phosphorylation. Yet PTEN, a PTPase-family phosphatase crucial to IR/Akt/ FoxO signaling, but acting downstream of IR/IGF1R by dephosphorylating phosphatidylinositol-30 ,40 ,50 -trisphosphate, was recently demonstrated to be inhibited by Zn ions and shown to be required for a Zn-induced modulation of Akt phosphorylation [38]. Based on the similar coordination properties of Zn(II) and intracellular Cu(I) [1], we hypothesize that these findings may also apply to copperinduced Akt activation in HepG2 cells. In contrast to Zn ions, however, copper ions are redox active in biological systems. Therefore, it remains to be elucidated both if copper ions inhibit PTEN in HepG2 cells at all and whether this is through coordinative occupation or through oxidation of the phosphatase’s active site. Acknowledgments This study was supported by the Natural Sciences and Engineering Research Council of Canada (NSERC, Discovery Grant RGPIN 402228-2011 to LOK). LOK acknowledges support by Canada Foundation for Innovation (CFI), Alberta Advanced Education and Technology (AET), and the Canada Research Chairs (CRC) Program. References [1] A. Barthel, E.A. Ostrakhovitch, P.L. Walter, A. Kampkötter, L.O. Klotz, Arch. Biochem. Biophys. 463 (2007) 175–182. [2] H.M. Korashy, A.O. El-Kadi, Free Radic. Biol. Med. 44 (2008) 795–806. [3] W. Wu, L.M. Graves, I. Jaspers, R.B. Devlin, W. Reed, J.M. Samet, Am. J. Physiol. 277 (1999) L924–L931. [4] M.D. Mattie, M.K. McElwee, J.H. Freedman, J. Mol. Biol. 383 (2008) 1008–1018. [5] E.A. Ostrakhovitch, M.R. Lordnejad, F. Schliess, H. Sies, L.O. Klotz, Arch. Biochem. Biophys. 397 (2002) 232–239. [6] P.L. Walter, A. Kampkötter, A. Eckers, A. Barthel, D. Schmoll, H. Sies, L.O. Klotz, Arch. Biochem. Biophys. 454 (2006) 107–113. [7] M. Monsalve, Y. Olmos, Curr. Drug Targets 12 (2011) 1322–1350. [8] D. Schmoll, K.S. Walker, D.R. Alessi, R. Grempler, A. Burchell, S. Guo, R. Walther, T.G. Unterman, J. Biol. Chem. 275 (2000) 36324–36333.

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