December 2015
Volume 56, Issue 13
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Biochemistry and Molecular Biology  |   December 2015
Inhibition of Protein Tyrosine Phosphatase 1B Improves IGF-I Receptor Signaling and Protects Against Inflammation-Induced Gliosis in the Retina
Author Affiliations & Notes
  • Ana I. Arroba
    Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), ISCIII, Madrid, Spain
    Instituto de Investigaciones Biomédicas “Alberto Sols” (CSIC/UAM), Madrid, Spain
  • Ángela M. Valverde
    Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), ISCIII, Madrid, Spain
    Instituto de Investigaciones Biomédicas “Alberto Sols” (CSIC/UAM), Madrid, Spain
  • Correspondence: Ángela M. Valverde, Instituto de Investigaciones Biomédicas “Alberto Sols” (CSIC/UAM), Madrid, Spain; [email protected]. Ana I. Arroba, Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBERDEM), ISCIII, Madrid, Spain; [email protected]
Investigative Ophthalmology & Visual Science December 2015, Vol.56, 8031-8044. doi:https://doi.org/10.1167/iovs.15-17234
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      Ana I. Arroba, Ángela M. Valverde; Inhibition of Protein Tyrosine Phosphatase 1B Improves IGF-I Receptor Signaling and Protects Against Inflammation-Induced Gliosis in the Retina. Invest. Ophthalmol. Vis. Sci. 2015;56(13):8031-8044. https://doi.org/10.1167/iovs.15-17234.

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      © ARVO (1962-2015); The Authors (2016-present)

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Abstract

Purpose: Insulin-like growth factor-I receptor (IGF-IR) signaling mediates retinal growth and survival and its failure may contribute to aggravate diabetic retinopathy (DR). Protein tyrosine phosphatase 1B (PTP1B) negatively modulates IGF-IR signaling, but its involvement in inflammation during DR remains unknown. We investigated whether PTP1B participates in the cross-talk between proinflammatory signaling pathways and IGF-IR–mediated signaling in the retina.

Methods: 661W photoreceptors or mouse retinal explants were treated with TNFα, IL6, and IL1β. Insulin-like growth factor-I receptor signaling cascade was evaluated in the absence or presence of PTP1B. db/db mice were used to test a PTP1B inhibitor in retinal gliosis.

Results: 661W retinal cells and retinal explants responded to IGF-I by inducing IGF-IR tyrosine (13-fold) and Akt phosphorylations (7- and 3-fold for serine 473 and threonine 308, respectively). Cytokines triggered early activation of stress kinases (c-jun [NH2] terminal kinase [JNK] and p38 MAPK), resulting in insulin receptor substrate 1 (IRS1) serine 307 phosphorylation that precedes its degradation. Pretreatment of 661W cells or retinal explants with cytokines upregulated PTP1B protein levels (1.45- and 4.5-fold, respectively), induced IRS1 degradation and decreased IGF-I–mediated IGF-IR/Akt phosphorylation. Silencing or deficiency in PTP1B ameliorated the negative effects of cytokines on IGF-IR signaling. Cytokines increased glial fibrillary acidic protein (GFAP) expression in retinal explants by 4.5-fold, this response being reduced by 2-fold with a PTP1B inhibitor. Protein tyrosine phosphatase 1B protein levels increased by 3-fold in retinas from db/db mice and its inhibition reduced gliosis.

Conclusions: Targeting PTP1B might be useful for modulating IGF-I effects in retinal cells during DR.

Diabetic retinopathy (DR) is a major cause of blindness globally and a leading cause of legal blindness in the working age population of the developing world.1,2 Diabetes affects the entire neurovascular unit of the retina, with gradual neurodegeneration, gliosis, neuroinflammation, compromise of the vascular blood–retinal barrier, edema, angiogenesis, and eventual fibrosis. Although neurodegeneration and inflammation have been implicated in the etiology of DR,35 diagnosis and treatment of this disease are mainly focused on the vascular abnormalities that ultimately occur. 
Type 2 diabetes mellitus (T2DM) is recognized as a low grade chronic inflammatory disease in which initially the infiltration of immune cells in adipose tissue generates a systemic proinflammatory milieu that negatively interferes with insulin signaling.6 Moreover, prolonged metabolic stress leads to a chronic state of overactivation of immune cells of the central nervous system (CNS), leading to neuroinflammation.7 Therefore, adipose-derived inflammatory mediators could be an important addition to local cytokines produced by CNS-resident microglia in boosting brain inflammation. A number of metabolic or molecular abnormalities associated to inflammation have been detected in retinas of diabetic animals, patients, or in retinal cells exposed to elevated concentrations of glucose8 raising the hypothesis that these defects might reflect an imbalance between pro- and anti-inflammatory cytokine expression.911 
Insulin and insulin-like growth factor-I receptors (IR and IGF-IR, respectively) belong to the tyrosine kinase receptor superfamily. Activation of both receptors stimulates tyrosine phosphorylation of insulin receptor substrates (IRS) that act as docking proteins engaging the receptors with intracellular signaling pathways. Among these, activation of phosphatidylinositol (PI) 3-kinase/Akt is essential for the cell survival.12,13 The IGF-I/IGF-IR system is expressed in retinal neurons14,15 and IGF-I is a proliferation and differentiation factor for the neural retina.16 
Recent evidences support the involvement of two recognized factors linking T2DM with neurodegeneration; the elevation of proinflammatory cytokines and the onset of insulin/IGF-I resistance.17 In T2DM, proinflammatory cytokines such as TNFα trigger phosphorylation of insulin receptor substrate 1 (IRS1) at serine 307 by stress kinases,18 which disrupts its interaction with the IR and blocks the intracellular actions of insulin in peripheral tissues. Likewise, excess of peripheral proinflammatory mediators, some of which can cross the blood–brain barrier, may trigger insulin/IGF-I resistance in the CNS, thereby weakening the neuroprotective signaling mediated by these molecules and this effect can contribute to the onset of neurodegenerative diseases.17 In this regard, it been demonstrated that IGF-I elicits neuroprotective effects in neurons exposed to several stressors including hypoxia-ischemia.19 
Protein tyrosine phosphatase 1B (PTP1B), an ubiquitously expressed phosphatase, has emerged as a relevant modulator of IR and IGF-IR signaling pathways by its ability to dephosphorylate tyrosines within the catalytic domain in both receptors.2022 In mice, deletion of the ptpn1 gene increased insulin sensitivity in liver and skeletal muscle and protects against obesity.23,24 In the retina, PTP1B is expressed in rod photoreceptor cells25 and also targets IGF-IR–mediated signaling.26 However, the modulation of the signaling pathways activated by the IGF-IR and the impact of PTP1B inhibition in the presence of a proinflammatory environment in the retina remains unknown and is the aim of the present study. 
Materials and Methods
Reagents
Fetal bovine serum (FBS) and culture media were from Invitrogen (Grand Island, NY, USA). Bovine serum albumin (BSA) was purchased from Sigma-Aldrich Corp. (St. Louis, MO, USA). IGF-I (human recombinant), IL1β, IL6, and TNFα were purchased from Preprotech (London, UK). Noncompetitive PTP1B inhibitor was 3-(3,5-dibromo-4-hydroxy-benzoyl)-2-ethyl-benzofuran-6-sulfonicacid-(4-(thiazol-2-ylsulfamyl)-phenyl)-amide (Catalog number 539741; Calbiochem, Darmstadt, Germany). Bradford reagent, acrylamide, immunoblot polyvinylidene difluoride (PVDF) membranes and chemiluminescent horseradish peroxidase (HRP) substrate were purchased from Bio-Rad (Madrid, Spain). 
Antibodies
Anti–IGF-IR (sc-713), anti–phospho-Akt1/2/3 (Ser473; sc-7985-R), anti–phospho-Akt1/2/3 (Thr308; sc-166646-R), anti-Akt1/2/3 (sc-8312), anti-JNK (sc-571), and anti-phospho p38 MAPK (Thr 180/Tyr182; sc-17852-R) antibodies were from Santa Cruz Biotechnology (Palo Alto, CA, USA). Anti-phospho IRS1 (Ser 307; 07-247), anti-p85α (06-195) and anti-PTP1B (05-1087, recognizes cleavaged PTP1B) and anti-PTP1B (07-088) antibodies were purchased from Merk-Millipore (Merck KGaA, Darmstadt, Germany). Anti–phospho-IGF-IR β (Tyr1135/1136; #3024), anti-phospho Akt (Ser473; #9272), anti-phospho JNK (Thr183/Tyr185) (#4668), and anti-phospho p38 MAPK (#9212) antibodies were purchased from Cell Signaling Technology (Danvers, MA, USA). Anti–α-tubulin (T-0198) antibody was from Sigma-Aldrich Corp. and anti–glial fibrillary acidic protein (GFAP) antibody (z0334) was obtained from DAKO (Glostrup, Denmark). 
Cell Culture
The 661W cell line derived from immortalized cone photoreceptors,27 provided by Muayyad Al-Ubaidi (University of Oklahoma, Norman, OK, USA), was maintained in Dulbecco's modified Eagle's medium (DMEM) supplemented with 10% (vol/vol) heat-inactivated FBS. For experiments, 661W cells were seeded at 75 × 103 cells/well in six-well plates. Confluent cells were grown in serum-free medium in the absence or presence of cytokines (CK; TNFα, IL6, and IL1β; 20 ng/mL each) for several time periods. In some experiments, after 24 hours of incubation with cytokines, IGF-I (10 nM) was added for the last 15 minutes. 
Retinal Explants
Ex vivo assays were performed with retinas from 10-week-old male C57BL/6 mice, db/+ and db/db mice, purchased from Charles River Laboratories (Charles River, Barcelona, Spain), or with age-matched PTP1B-deficient (PTP1B−/−) mice.26 Mice were maintained in light/dark (12-hours light/12-hours dark), temperature (22°C), and humidity-controlled rooms, and fed ad libitum with free access to water. All animal experimentation followed recommendations of the Federation of European Laboratory Animal Science Associations (FELASA) on health monitoring in accordance with the regulations of the Association for Research in Vision and Ophthalmology (ARVO). Animals were killed by cervical dislocation and eyes were enucleated. The lens, anterior segment, vitreous body, retinal pigment epithelium, and sclera were removed and the retina was cultured in R16 medium (provided by PA Ekstrom, Lund University, Sweden) with no additional serum. Retinas were stimulated with cytokines (CK; TNFα, IL6, and IL1β; 20 ng/mL each) and/or IGF-I (20 nM) at the time periods indicated in the figure legends. 
Quantitative Real-Time PCR Analysis
Total RNA was extracted with Trizol (Fisher Scientific-USA, Waltham, MA, USA) and reverse transcribed using High-Capacity cDNA Reverse Transcription Kit (ThermoFisher Scientific, Madrid, Spain). Quantitative real-time PCR (qPCR) was performed with an ABI 7900 sequence detector (ThermoFisher Scientific). The primer-probe sets for mouse ptpn1 and 18s were purchased as predesigned TaqMan gene expression (Life Technologies; catalog numbers Mm_00448427_m1 and Mm 04277571, respectively). The PCR cycles were 95°C for 10 minutes, 40 cycles at 95°C for 15 seconds and 60°C for 60 seconds following the manufacturer's instructions (Applied Biosystems, Madrid, Spain). 
Transient Transfection With siRNA
siRNA oligonucleotides were synthesized by Dharmacon RNAi Technologies (Fisher Scientific-USA). Targeted sequences for ptpn1 were: 5′GGAGAAAGGCUCGUUAAAA3′; 5′GACCACAGUCGGAUUAAAU3′; 5′GGUUUGAAGUUGACACUAA3′; 5′UCGCGAAGCUUCCUAAGAA3′ (siGENOME SMART pool, mouse ptpn1). 661W cells and retinal explants were transfected with scrambled (siGENOME RISC-free Control siRNA; catalog number D-001220-01; Fisher Scientific-USA) or PTP1B siRNAs at 25 nM concentration following manufacturer's instructions. After 36 hours, transfected cells and retinal explants were treated with cytokines (CK; 20 ng/mL) for 24 hours, serum-starved for 2 hours and further stimulated with IGF-I (10 nM) for 15 minutes. Scrambled or PTP1B siRNA oligos were present during treatment with cytokines and IGF-I. 
Western Blotting
Proteins were resolved using denaturing SDS-PAGE, and transferred to PVDF membranes (Bio-Rad).26 Membranes were blocked using 5% nonfat dried milk or 3% BSA in 10 mM Tris-HCl, 150 mM NaCl, pH 7.5 (TBS), and incubated overnight with antibodies in 0.05% Tween-20-TBS. Immunoreactive bands were visualized using the enhanced chemioluminiscence reagent (Bio-Rad). Levels of phosphorylated proteins (pIGF-IR and pAkt) were normalized by levels of corresponding total (IGF-IR and Akt) proteins that were used as loading controls. These results are expressed as fold change of IGF-I stimulation related to each basal. Levels of PTP1B were normalized by α-tubulin. 
PTP1B Enzymatic Activity
PTP1B phosphatase activity was determined by measuring phosphate release using a synthetic monophosphotyrosyl-containing peptide and the malachite green assay (Ref. 17-125; Millipore) as described.28 
Histologic Analysis and Immunofluorescence
Retinal explants were fixed in 4% paraformaldehyde for 8 hours, washed in TBS containing 0.1% (wt/vol) BSA and 0.1% (vol/vol) Triton X-100 (this buffer was used for all subsequent washes) and blocked and permeated for 2 hours in TBS containing 3% (wt/vol) BSA and 1% (vol/vol) Triton X-100. The samples were incubated overnight in a humid chamber at 4°C with rabbit anti-GFAP antibody in blocking solution. Retinal explants were washed and incubated for 90 minutes with anti-rabbit immunoglobulin antibody conjugated to Alexa 488 (Molecular Probes, Eugene, OR, USA), washed, and mounted with mounting medium (Fluoromount G). Staining was observed with a laser confocal microscope LSM710 (Carl Zeiss Microscopy GmbH, Göttingen, Germany). For quantification of GFAP immunostaining, mean fluorescence density was measured in five fields per retina using the ImageJ software (http://imagej.nih.gov/ij/; provided in the public domain by the National Institutes of Health, Bethesda, MD, USA). 
Statistical Analysis
Densitometry of the Western blots was performed using the ImageJ program. Values in all graphs represented the mean ± SEM. Statistical tests were performed using SPSS 21.0 for Windows (SPSS, Inc., IBM, Armonk, NY, USA). Data were analyzed by 1- or 2-way ANOVA followed by Bonferroni t-test or by paired t-test when comparisons were among two groups. Differences were considered significant at P ≤ 0.05. 
Results
IGF-I Induces Phosphorylation of the IGF-IR and Akt in Retinal Cells
Insulin-like growth factor-I and its receptor (IGF-IR) are expressed in the inner retina,14,15 and retinal microvascular cells produce,29 and respond to IGF-I in vitro.30 Because activation of the IGF-IR/Akt signaling cascade protects mammalian cell types against death signals,31 we characterized the activation of this pathway in 661W cells that expressed cone photoreceptor markers.27 As shown in Figures 1A and 1B, in 661W photoreceptors phosphorylation of the IGF-IR at tyrosines 1135/1136 within the catalytic domain and Akt at threonine 308 occurred in a dose-dependent manner; being maximal effect elicited at 10 nM (13-fold for IGF-IR tyrosine phosphorylation and 7-fold for Akt threonine 308 phosphorylation). Maximal phosphorylation of Akt at serine 473 (3-fold), which was mediated by the mTORC2 complex,32 was detected at 0.1 nM. These results indicate that 661W retinal cells respond to IGF-I and agree with the activation of IGF-IR/Akt signaling by IGF-I in retinal explants.26 
Figure 1
 
Insulin-like growth factor-IR–mediated Akt signaling in 661W retinal cells. Confluent 661W cells were serum-starved for 2 hours and then stimulated with various doses of IGF-I for 15 minutes. Protein extracts (30 μg) were separated by SDS-PAGE and analyzed by Western blot with the anti-phospho IGF-IR (Tyr1135/1136), anti–IGF-IR, anti-phospho Akt (Ser473), anti-phospho Akt (Thr308), and anti-Akt antibodies. (A) Representative autoradiograms are shown (n = 3 independent experiments). (B) The blots were quantitated by scanning densitometry. Levels of phosphorylated IGF-IR and Akt were normalized by their corresponding levels of total IGF-I or Akt protein, respectively, that were used as loading control. Results are expressed as fold increase of IGF-I stimulation and are means ± SEM. Data were analyzed by 1-way ANOVA followed by Bonferroni t-test. Comparisons were *†‡P ≤ 0.05 IGF-I stimulation versus their corresponding nonstimulated basal (B).
Figure 1
 
Insulin-like growth factor-IR–mediated Akt signaling in 661W retinal cells. Confluent 661W cells were serum-starved for 2 hours and then stimulated with various doses of IGF-I for 15 minutes. Protein extracts (30 μg) were separated by SDS-PAGE and analyzed by Western blot with the anti-phospho IGF-IR (Tyr1135/1136), anti–IGF-IR, anti-phospho Akt (Ser473), anti-phospho Akt (Thr308), and anti-Akt antibodies. (A) Representative autoradiograms are shown (n = 3 independent experiments). (B) The blots were quantitated by scanning densitometry. Levels of phosphorylated IGF-IR and Akt were normalized by their corresponding levels of total IGF-I or Akt protein, respectively, that were used as loading control. Results are expressed as fold increase of IGF-I stimulation and are means ± SEM. Data were analyzed by 1-way ANOVA followed by Bonferroni t-test. Comparisons were *†‡P ≤ 0.05 IGF-I stimulation versus their corresponding nonstimulated basal (B).
Retinal Cells Respond to Proinflammatory Cytokines by the Activation of Stress Kinases
Because proinflammatory cytokines were components of the diabetic milieu in the retina,33 we inspected the sensitivity of retinal cells to the activation of stress kinases in response to proinflammatory cytokines. A mixture of proinflammatory cytokines (TNFα, IL6, and IL1β) was used to stimulate 661W cells and phosphorylations of c-jun (NH2) terminal kinase (JNK) and p38 mitogen-activated protein kinase (MAPK) were analyzed. As shown in Figures 2A and 2B, phosphorylation of JNK and p38 MAPK was very rapid and peaked at 15 and 30 minutes, respectively, after addition of cytokines. A 14- and 2.5-fold increase in JNK and p38 MAPK phosphorylations, respectively, were found at these time periods. 
Figure 2
 
A combination of proinflammatory cytokines (CK) induced the activation of stress kinases and the phosphorylation of IRS1 at serine 307 in retinal cells. (A) 661W photoreceptors and (C) mouse retinal explants were stimulated with a combination of proinflammatory cytokines containing TNFα, IL6, and IL1β (CK; 20 ng/mL each) at the indicated time-periods. Protein extracts (50 μg) were separated by SDS-PAGE and the phosphorylation of IRS1 (Ser307), JNK (Thr183/Tyr185), and p38 MAPK (Thr180/Tyr182) was analyzed by Western blot with the corresponding antibodies. Total levels of IRS1, JNK, and p38 MAPK were also analyzed. Representative autoradiograms are shown (n = 3 independent experiments). In (B) and (D) the results are means ± SEM. Data were analyzed by 1-way ANOVA followed by Bonferroni t-test. Comparisons were *†‡P ≤ 0.05 cytokines (CK) versus their corresponding nonstimulated basal (B).
Figure 2
 
A combination of proinflammatory cytokines (CK) induced the activation of stress kinases and the phosphorylation of IRS1 at serine 307 in retinal cells. (A) 661W photoreceptors and (C) mouse retinal explants were stimulated with a combination of proinflammatory cytokines containing TNFα, IL6, and IL1β (CK; 20 ng/mL each) at the indicated time-periods. Protein extracts (50 μg) were separated by SDS-PAGE and the phosphorylation of IRS1 (Ser307), JNK (Thr183/Tyr185), and p38 MAPK (Thr180/Tyr182) was analyzed by Western blot with the corresponding antibodies. Total levels of IRS1, JNK, and p38 MAPK were also analyzed. Representative autoradiograms are shown (n = 3 independent experiments). In (B) and (D) the results are means ± SEM. Data were analyzed by 1-way ANOVA followed by Bonferroni t-test. Comparisons were *†‡P ≤ 0.05 cytokines (CK) versus their corresponding nonstimulated basal (B).
We also analyzed these responses in the whole retina using retinal explants that preserved the sensitivity to extracellular stimuli26 and found maximal JNK (4-fold) and p38 MAPK (12-fold) phosphorylations at 1 and 2 hours, respectively (Figs. 2C, 2D). 
Serine phosphorylation of IRS1, an immediate downstream target of the IGF-IR, precedes its ubiquitination and proteasomal degradation.34,35 Among potential IRS1 serine kinases, JNK has been extensively studied in insulin resistant states.36 Treatment with cytokines increased IRS1 serine 307 phosphorylation in 661W cells by 3.5-fold and, importantly, similar effect occurred in retinal explants where cytokines increased this response by 15-fold (Figs. 2A–D). 
Proinflammatory Cytokines Negatively Interfered With IGF-IR/Akt Signaling in Retinal Cells
Because proinflammatory cytokines trigger early and long-term signaling pathways that negatively modulate IGF-IR–mediated signaling,37 we analyzed this cross-talk in retinal cells. 661W cells were pretreated with cytokines for 24 hours, serum-starved for 2 hours, and further stimulated with IGF-I (10 nM) for 15 minutes. This prolonged treatment with cytokines did not induce death of 661W cells (results not shown). Insulin-like growth factor-I–induced IGF-IR tyrosine phosphorylation decreased by 6.5-fold in 661W cells pretreated with cytokines as compared with nonpretreated cells (CKI versus I; Fig. 3A). Downstream the IGF-IR, IRS1 levels significantly decreased (0.5-fold) in the presence of cytokines, reflecting its proteosomal degradation (Fig. 3B). Likewise, the response to IGF-I in the phosphorylation of Akt (threonine 308 and serine 473) was decreased by 7.5- and 3.5-fold, respectively, in cells pretreated with cytokines compared with nonpretreated cells (CKI versus I; Fig. 3C). 
Figure 3
 
Proinflammatory cytokines activated signaling pathways that negatively modulated IGF-IR–mediated signaling. 661W cells (AC) and retinal explants (D, E) were treated with cytokines (CK; 20 ng/mL) for 24 hours, serum-starved for 2 hours, and further stimulated with IGF-I (I; 10 nM) for 15 minutes (661W retinal cells) or IGF-I (I; 20 nM) for 30 minutes (retinal explants). In (B) (661W cells) and (E) (retinal explants) the treatment was 24 hours with cytokines. Protein extracts (50 μg) were separated by SDS-PAGE and analyzed by Western blot with antibodies against phospho–IGF-IR (Tyr1135/1366), IGF-IR, phospho-Akt (Ser473), phospho-Akt (Thr308), Akt, IRS1, and α-tubulin. Representative autoradiograms are shown (n = 6 independent experiments). The blots were quantitated by scanning densitometry. Levels of phosphorylated proteins (pIGF-IR and pAkt) were normalized by levels of their corresponding total IGF-IR and Akt protein that were used as loading controls. These results are expressed as fold change of IGF-I stimulation related to each basal. Alpha-tubulin was used as a loading control for IRS1. The results are means ± SEM. Data were analyzed by 1- or 2-way ANOVA followed by Bonferroni t-test. In (A, C, D) comparisons were *P ≤ 0.05 IGF-I (I) versus nonstimulated basal (B); †P ≤ 0.05 cytokines plus IGF-I (CKI) versus IGF-I (I). In (B) and (E) results are means ± SEM. Data were analyzed by paired t-test. Comparisons were *P ≤ 0.05 cytokines (CK) versus nonstimulated basal (B).
Figure 3
 
Proinflammatory cytokines activated signaling pathways that negatively modulated IGF-IR–mediated signaling. 661W cells (AC) and retinal explants (D, E) were treated with cytokines (CK; 20 ng/mL) for 24 hours, serum-starved for 2 hours, and further stimulated with IGF-I (I; 10 nM) for 15 minutes (661W retinal cells) or IGF-I (I; 20 nM) for 30 minutes (retinal explants). In (B) (661W cells) and (E) (retinal explants) the treatment was 24 hours with cytokines. Protein extracts (50 μg) were separated by SDS-PAGE and analyzed by Western blot with antibodies against phospho–IGF-IR (Tyr1135/1366), IGF-IR, phospho-Akt (Ser473), phospho-Akt (Thr308), Akt, IRS1, and α-tubulin. Representative autoradiograms are shown (n = 6 independent experiments). The blots were quantitated by scanning densitometry. Levels of phosphorylated proteins (pIGF-IR and pAkt) were normalized by levels of their corresponding total IGF-IR and Akt protein that were used as loading controls. These results are expressed as fold change of IGF-I stimulation related to each basal. Alpha-tubulin was used as a loading control for IRS1. The results are means ± SEM. Data were analyzed by 1- or 2-way ANOVA followed by Bonferroni t-test. In (A, C, D) comparisons were *P ≤ 0.05 IGF-I (I) versus nonstimulated basal (B); †P ≤ 0.05 cytokines plus IGF-I (CKI) versus IGF-I (I). In (B) and (E) results are means ± SEM. Data were analyzed by paired t-test. Comparisons were *P ≤ 0.05 cytokines (CK) versus nonstimulated basal (B).
To demonstrate the physiological relevance of these data, mouse retinas were incubated ex vivo with cytokines for 24 hours and then stimulated with IGF-I (20 nM) for 30 minutes. Cell death was not detected in retinal explants treated with cytokines (results not shown). Pretreatment with cytokines reduced the response to IGF-I in inducing IGF-IR tyrosine phosphorylation at basal levels (CKI versus I) and also reduced by 1.75 fold Akt phosphorylation at serine 473 as compared with explants that did not receive proinflammatory stimuli (CKI versus I; Fig. 3D). Notably, in retinal explants the treatment with cytokines increased basal Akt phosphorylation by 5.5 fold (CK versus B), but this response was not further increased by IGF-I stimulation, reflecting the lack of sensitivity to IGF-I. Degradation of IRS1 was found in retinal explants cultured in the presence of cytokines compared with the basal condition (0.5-fold decrease in CK versus B; Fig. 3E). 
Modulation of PTP1B Expression and Enzymatic Activity in Retinal Photoreceptors and Retinal Explants Under Proinflammatory Conditions
We have demonstrated that PTP1B is elevated in the retina of diabetic mice and the modulation of IGF-IR tyrosine phosphorylation by this phosphatase in retinal explants.26 Based on that, we analyzed the effect of the proinflammatory milieu on PTP1B expression in photoreceptors. As Figure 4A showed, PTP1B mRNA was elevated by 2.5-fold in 661W cells treated with cytokines. Moreover, cytokines induced the cleavage of the protein with a similar pattern reported in platelets38 and also increased PTP1B protein levels by 1.5-fold (Fig. 4B) and enzymatic activity by 2-fold reflecting the activation of the phosphatase activity associated to this cleavage38 (Fig. 4C). In agreement with data in peripheral tissues,39 PTP1B mRNA and protein expression were also increased by 3.8- and 4.5-fold, respectively, in retinal explants cultured with cytokines (Figs. 4D, 4E). 
Figure 4
 
Protein tyrosine phosphatase 1B expression is upregulated in retinal cells and retinal explants cultured under proinflammatory conditions. (AC) For 24 hours, 661W cells were treated with cytokines (CK; 20 ng/mL). (A) Protein tyrosine phosphatase 1B mRNA levels were determined by real-time PCR. Used as a control was 18S RNA. Results are expressed as 2−ΔCt. (B) Protein extracts (50 μg) were separated by SDS-PAGE and analyzed by Western blot with the anti-PTP1B antibody (Ref. 05-1087; Millipore). Alpha-tubulin was used as a loading control. Representative autoradiograms are shown. (C) Protein tyrosine phosphatase 1B enzymatic activity was analyzed as described in Materials and Methods. The results of PTP1B enzymatic activity are expressed as picomoles of released phosphate/min/g protein. The results are means ± SEM (n = 5 independent experiments). Data were analyzed by Student's paired t-test; *P ≤ 0.05 cytokines (CK) versus nonstimulated basal (B). Retinal explants were treated with cytokines (CK; 20 ng/mL) for 24 hours. (D) Protein tyrosine phosphatase 1B mRNA levels were determined by real-time PCR. Used as a control was 18S RNA. Results are expressed as 2−ΔCt. (E) Protein extracts (50 μg) were separated by SDS-PAGE and analyzed by Western blot with the anti-PTP1B antibody (Ref. 05-1087; Millipore). Alpha-tubulin was used as a loading control. The results are expressed as fold change and are means ± SEM (n = 12 independent retinas per condition). Data were analyzed by Student's paired t-test. Comparisons were *P ≤ 0.05 cytokines (CK) versus nonstimulated basal (B).
Figure 4
 
Protein tyrosine phosphatase 1B expression is upregulated in retinal cells and retinal explants cultured under proinflammatory conditions. (AC) For 24 hours, 661W cells were treated with cytokines (CK; 20 ng/mL). (A) Protein tyrosine phosphatase 1B mRNA levels were determined by real-time PCR. Used as a control was 18S RNA. Results are expressed as 2−ΔCt. (B) Protein extracts (50 μg) were separated by SDS-PAGE and analyzed by Western blot with the anti-PTP1B antibody (Ref. 05-1087; Millipore). Alpha-tubulin was used as a loading control. Representative autoradiograms are shown. (C) Protein tyrosine phosphatase 1B enzymatic activity was analyzed as described in Materials and Methods. The results of PTP1B enzymatic activity are expressed as picomoles of released phosphate/min/g protein. The results are means ± SEM (n = 5 independent experiments). Data were analyzed by Student's paired t-test; *P ≤ 0.05 cytokines (CK) versus nonstimulated basal (B). Retinal explants were treated with cytokines (CK; 20 ng/mL) for 24 hours. (D) Protein tyrosine phosphatase 1B mRNA levels were determined by real-time PCR. Used as a control was 18S RNA. Results are expressed as 2−ΔCt. (E) Protein extracts (50 μg) were separated by SDS-PAGE and analyzed by Western blot with the anti-PTP1B antibody (Ref. 05-1087; Millipore). Alpha-tubulin was used as a loading control. The results are expressed as fold change and are means ± SEM (n = 12 independent retinas per condition). Data were analyzed by Student's paired t-test. Comparisons were *P ≤ 0.05 cytokines (CK) versus nonstimulated basal (B).
Reduction of PTP1B in Retinal Cells Protected Against Inflammation-Mediated Inhibition of IGF-IR Signaling
We have demonstrated that PTP1B inhibition restored hepatic insulin sensitivity in several in vivo models of insulin resistance.28,40,41 On that basis, we investigated the effect of cytokines on IGF-IR–mediated signaling in retinal cells with reduced PTP1B expression using siRNA. Protein tyrosine phosphatase 1B protein content was decreased by almost 90% in 661W cells transfected with PTP1B siRNA (Fig. 5A). Then, we analyzed IGF-IR–mediated signaling in the presence of cytokines and we found that PTP1B silencing maintained IGF-IR tyrosine phosphorylation and Akt (serine 473) phosphorylation in the presence of cytokines compared with the reduced responses of the control (scrambled) condition. In cells transfected with scrambled siRNA, IGF-I–mediated IGF-IR phosphorylation was reduced by 4-fold when they were pretreated with cytokines (CKI SC versus I SC; Figs. 5B, 5C). Moreover, in cells transfected with PTP1B siRNA, IGF-I–mediated IGF-IR phosphorylation in the presence of cytokines was 5-fold higher than the response of this condition in the scrambled siRNA treatment (CKI siRNA PTP1B versus CKI SC). Regarding Akt, IGF-I–mediated phosphorylation at serine 473 was reduced by 7.6-fold when cells were pretreated with cytokines (CKI SC versus I SC). By contrast, in cells transfected with PTP1B siRNA this response was 8.2-fold increase compared with this condition in the scrambled siRNA treatment (CKI siRNA PTP1B versus CKI SC). 
Figure 5
 
Reduction of PTP1B levels by siRNA in retinal cells protected against inflammation-mediated inhibition of IGF-IR signaling. (A) Efficiency of PTP1B siRNA in decreasing PTP1B levels in 661W cells. (B, C) For 36 hours, 661W cells were transfected with scrambled or PTP1B siRNA oligonucleotides and transfected cells were treated with cytokines (CK; 20 ng/mL) for 24 hours, serum-starved for 2 hours and further stimulated with IGF-I (10 nM) for 15 minutes. (D) Efficiency of PTP1B siRNA in decreasing PTP1B in retinal explants. (E, F) Retinal explants were treated with siRNAs and stimulated as described for 661W cells. (G) Retinal explants from PTP1B−/− mice were stimulated with cytokines and IGF-I as indicated above. Protein extracts (50 μg) were separated by SDS-PAGE and analyzed by Western blot with the corresponding antibodies: anti-PTP1B (Ref. 07-088; Millipore), anti-phospho IGF-IR (Tyr 1135/1136), anti-IGF-IR, anti-phospho Akt (Ser473), anti-Akt, and anti–α-tubulin. Representative autoradiograms are shown (n = 5 independent experiments or retinas per experimental condition). Autoradiograms were quantitated by scanning densitometry. Levels of phosphorylated proteins (pIGF-IR and pAkt) were normalized by levels of corresponding total (IGF-IR and Akt) proteins that were used as loading controls. These results are expressed as fold change of IGF-I stimulation related to each basal. Levels of PTP1B were normalized by α-tubulin. The results are means ± SEM. In (A) and (D), data were analyzed by paired t-test and comparisons were *P ≤ 0.05 siRNA PTP1B versus scrambled siRNA (SC). In (C) and (F), data were analyzed by 1- or 2-way ANOVA followed by Bonferroni t-test. Comparisons were *P ≤ 0.05 CKI siRNA PTP1B versus CKI SC.
Figure 5
 
Reduction of PTP1B levels by siRNA in retinal cells protected against inflammation-mediated inhibition of IGF-IR signaling. (A) Efficiency of PTP1B siRNA in decreasing PTP1B levels in 661W cells. (B, C) For 36 hours, 661W cells were transfected with scrambled or PTP1B siRNA oligonucleotides and transfected cells were treated with cytokines (CK; 20 ng/mL) for 24 hours, serum-starved for 2 hours and further stimulated with IGF-I (10 nM) for 15 minutes. (D) Efficiency of PTP1B siRNA in decreasing PTP1B in retinal explants. (E, F) Retinal explants were treated with siRNAs and stimulated as described for 661W cells. (G) Retinal explants from PTP1B−/− mice were stimulated with cytokines and IGF-I as indicated above. Protein extracts (50 μg) were separated by SDS-PAGE and analyzed by Western blot with the corresponding antibodies: anti-PTP1B (Ref. 07-088; Millipore), anti-phospho IGF-IR (Tyr 1135/1136), anti-IGF-IR, anti-phospho Akt (Ser473), anti-Akt, and anti–α-tubulin. Representative autoradiograms are shown (n = 5 independent experiments or retinas per experimental condition). Autoradiograms were quantitated by scanning densitometry. Levels of phosphorylated proteins (pIGF-IR and pAkt) were normalized by levels of corresponding total (IGF-IR and Akt) proteins that were used as loading controls. These results are expressed as fold change of IGF-I stimulation related to each basal. Levels of PTP1B were normalized by α-tubulin. The results are means ± SEM. In (A) and (D), data were analyzed by paired t-test and comparisons were *P ≤ 0.05 siRNA PTP1B versus scrambled siRNA (SC). In (C) and (F), data were analyzed by 1- or 2-way ANOVA followed by Bonferroni t-test. Comparisons were *P ≤ 0.05 CKI siRNA PTP1B versus CKI SC.
In mouse retinal explants, PTP1B siRNA efficiently reduced almost 70% PTP1B protein levels (Fig. 5D) and improved IGF-IR/Akt-mediated signaling in the presence of cytokines. Insulin-like growth factor-I–mediated Akt phosphorylation at serine 473 was reduced by 3.6-fold when retinal explants were pretreated with cytokines (CKI SC versus I SC; Figs. 5E, 5F). In retinal explants transfected with PTP1B siRNA, IGF-I–mediated Akt serine 473 phosphorylation in the presence of cytokines was 13.3-fold higher than the response of this condition in the scrambled siRNA treatment (CKI siRNA PTP1B versus CKI SC). 
To reinforce data obtained with siRNA in a genetic modified mouse model, we prepared retinal explants from PTP1B-deficient mice (PTP1B−/−) as previously reported.26 Interestingly, the treatment with cytokines did not reduce the response to IGF-I regarding Akt phosphorylation in retinas from PTP1B−/− mice (Fig. 5G). 
We did not detect cell death in either 661W cells or retinal explants transfected with siRNA (results not shown). 
Pharmacologic Inhibition of PTP1B Reduced Reactive Gliosis in Retinal Explants Treated With Cytokines
Neurodegenerative diseases of the retina, including AMD, DR, and glaucoma involve chronic and dysregulated neuroinflammation and reactive gliosis.42,43 To get insight into the benefit of PTP1B inhibition in the whole retina under proinflammatory conditions, we evaluated the effect of cytokines on reactive gliosis in the ex vivo model of mouse retinal explants. Treatment with cytokines for 24 hours increased GFAP expression analyzed by Western blot by 4.5-fold (Fig. 6A) and also increased GFAP staining (10-fold) in live retinal explants (Fig. 6B) indicating that, in the retina, cytokine-mediated reactive gliosis concurred with disruption of the IGF-IR–mediated signaling cascade. We also analyzed the effect of pharmacologic inhibition of PTP1B in reactive gliosis. Cotreatment with cytokines and the PTP1B inhibitor used at 10 μM as described44 decreased by 2-fold GFAP protein levels in mouse retinal explants (Fig. 6A). 
Figure 6
 
Pharmacologic inhibition of PTP1B reduced reactive gliosis induced by proinflammatory cytokines in retinal explants. Whole retinas were obtained from 10-week-old mice and cultured with cytokines (CK; 20 ng/mL) for 24 hours in the presence or absence of the PTP1B inhibitor (10 μM). (A) Protein extracts (50 μg) were separated by SDS-PAGE and analyzed by Western blot with the corresponding antibodies against GFAP and α-tubulin that was used as a loading control. Representative autoradiograms are shown. Autoradiograms corresponding to GFAP levels were quantitated by scanning densitometry. The results are means ± SEM (n = 5 retinas per experimental condition). Data were analyzed by 1-way ANOVA followed by Bonferroni t-test. Comparisons were *P ≤ 0.05 cytokines (CK) versus basal (B); †P ≤ 0.05 CK plus PTP1B inhibitor versus CK. (B) Whole retinas were obtained from 10-week-old mice, cultured as described in (A) an immunostaining for GFAP (green) was carried out. Five retinas per experimental condition were analyzed. Representative confocal images are shown. Quantification of immunofluorescence was performed as described in Methods. Data were analyzed by paired t-test. Comparisons were *P ≤ 0.05 CK versus basal (B).
Figure 6
 
Pharmacologic inhibition of PTP1B reduced reactive gliosis induced by proinflammatory cytokines in retinal explants. Whole retinas were obtained from 10-week-old mice and cultured with cytokines (CK; 20 ng/mL) for 24 hours in the presence or absence of the PTP1B inhibitor (10 μM). (A) Protein extracts (50 μg) were separated by SDS-PAGE and analyzed by Western blot with the corresponding antibodies against GFAP and α-tubulin that was used as a loading control. Representative autoradiograms are shown. Autoradiograms corresponding to GFAP levels were quantitated by scanning densitometry. The results are means ± SEM (n = 5 retinas per experimental condition). Data were analyzed by 1-way ANOVA followed by Bonferroni t-test. Comparisons were *P ≤ 0.05 cytokines (CK) versus basal (B); †P ≤ 0.05 CK plus PTP1B inhibitor versus CK. (B) Whole retinas were obtained from 10-week-old mice, cultured as described in (A) an immunostaining for GFAP (green) was carried out. Five retinas per experimental condition were analyzed. Representative confocal images are shown. Quantification of immunofluorescence was performed as described in Methods. Data were analyzed by paired t-test. Comparisons were *P ≤ 0.05 CK versus basal (B).
Pharmacologic Inhibition of PTP1B Reduced Reactive Gliosis in Retinal Explants From db/db Mice
It has been reported that diabetic db/db mice presented systemic inflammation45 and reactive gliosis in the retina.3 On that basis, we analyzed PTP1B expression in retinas of these mice. Protein tyrosine phosphatase 1B mRNA and protein levels were increased by 1.8- and 3-fold, respectively, in retinas from db/db mice compared with the lean db/+ controls (Figs. 7A, 7B). Finally, we evaluated the efficacy of the PTP1B inhibitor against the reactive gliosis of db/db mice.3 As depicted in Figure 7C, GFAP immunostaining was decreased by 0.4-fold in retinal explants from db/db mice cultured with the PTP1B inhibitor for 24 hours. 
Figure 7
 
Pharmacologic inhibition of PTP1B reduced reactive gliosis in db/db mice. Whole retinas were obtained from 10-week-old db/+ and db/db mice. (A) Protein tyrosine phosphatase 1B mRNA levels were determined by real-time PCR. As a control, 18S RNA was used. Results are expressed as 2−ΔCt. (B) Protein extracts (50 μg) were separated by SDS-PAGE and analyzed by Western blot with the anti-PTP1B antibody (Ref. 05-1087; Millipore). Alpha-tubulin was used as a loading control. Representative autoradiograms are shown. The results are means ± SEM (n = 12 independent retinas per condition). Data were analyzed by Student's paired t-test. Comparisons were *P ≤ 0.05 db/db versus db/+. (C) Immunostaining for GFAP (green) was carried out in retinas from db/+ and db/db mice (upper panels) and in retinas from db/db mice were cultured for 24 hours in the presence or absence of the PTP1B inhibitor (10 μM; lower panels). Five retinas per experimental condition were analyzed. Representative images are shown. Quantification of immunofluorescence was performed as described in Methods. Data were analyzed by paired t-test. Comparisons were *P ≤ 0.05 db/db versus db/+ and †P ≤ 0.05 db/db PTP1B inhibitor versus db/db basal.
Figure 7
 
Pharmacologic inhibition of PTP1B reduced reactive gliosis in db/db mice. Whole retinas were obtained from 10-week-old db/+ and db/db mice. (A) Protein tyrosine phosphatase 1B mRNA levels were determined by real-time PCR. As a control, 18S RNA was used. Results are expressed as 2−ΔCt. (B) Protein extracts (50 μg) were separated by SDS-PAGE and analyzed by Western blot with the anti-PTP1B antibody (Ref. 05-1087; Millipore). Alpha-tubulin was used as a loading control. Representative autoradiograms are shown. The results are means ± SEM (n = 12 independent retinas per condition). Data were analyzed by Student's paired t-test. Comparisons were *P ≤ 0.05 db/db versus db/+. (C) Immunostaining for GFAP (green) was carried out in retinas from db/+ and db/db mice (upper panels) and in retinas from db/db mice were cultured for 24 hours in the presence or absence of the PTP1B inhibitor (10 μM; lower panels). Five retinas per experimental condition were analyzed. Representative images are shown. Quantification of immunofluorescence was performed as described in Methods. Data were analyzed by paired t-test. Comparisons were *P ≤ 0.05 db/db versus db/+ and †P ≤ 0.05 db/db PTP1B inhibitor versus db/db basal.
Discussion
In chronic diseases of the retina linked to neuroinflammation, retinal macroglia, and microglia populations demonstrate activation and gliosis,46,47 which contribute to neurodegeneration in the neighbor retinal cells and, ultimately, in vision loss.4,48 Thus, unravelling the molecular mechanisms that confer protection to retinal cells against chronic neuroinflammation, as well as elucidating their therapeutic modulation, is of key importance. So far, current treatments for DR are applicable only at advanced stages and are associated with adverse effects. Therefore, new pharmacologic treatments aimed to combat retinal neuroinflammation associated to DR are urgently needed. This study provides evidence of the beneficial effect of targeting PTP1B in the retina in the IGF-IR/Akt survival signaling pathway and reactive gliosis. 
Although IGF-I has been recognized for its peripheral effects in growth and metabolism, it has a role in the CNS as a neurotrophic peptide triggering prosurvival signaling pathways responsible for activation of antiapoptotic cascades, enhancement of nerve growth, and promotion of synaptic plasticity.49 Moreover, IGF-IR signaling reduces neuroinflammation-dependent sensitivity of neurons to the proparkinsonian neurotoxin 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).50 In the retina, early intervention with systemic IGF-I in diabetic rats inhibits neuroretinal cell death.51 However, at the molecular level the impact of the chronic inflammatory environment, which is present in the retina in insulin resistance and T2DM,52 in the IGF-IR signaling in retinal cells has not been investigated in depth. Taking into account these previous findings, this study has identified for the first time a negative cross-talk between signaling pathways activated by the proinflammatory milieu and the IGF-IR/Akt neuroprotective pathway in photoreceptors that are major contributors to diabetes-induced local inflammation in the retina.53,54 We found that 661W cells are direct targets of proinflammatory cytokines as manifested by a rapid phosphorylation of JNK and p38 MAPK similar to that of peripheral cells during obesity.40,55,56 Because a previous report described that activation of JNK in response to light preceded apoptosis in 661W cells,57 our results suggest that under conditions in which chronic inflammation reaches the retina, activation of stress kinases occur as it does in peripheral insulin-sensitive cells. In this regard, the feedback mechanism mediated by JNK responsible of the early phosphorylation of IRS1 at serine 307 that targets it for proteasomal degradation35 was found in photoreceptors and retinal explants cultured with proinflammatory cytokines. We have successfully used retinal organotypic cultures to characterize pathologic cell death during retinal degeneration.58 Herein, we made use of these cultures for elucidating the signaling pathways deregulated in DR. Considering these previous findings, long-term (24 hours) exposure of retinal cells and retinal explants to cytokines decreased IGF-IR tyrosine phosphorylation in response to IGF-I and triggered the degradation of IRS1. Intriguing, increased Akt phosphorylation was found in retinal explants treated with cytokines. This might result of compensatory mechanisms that could alter the expression or activity of specific serine phosphatases attempting to counteract the deleterious effects of cytokines. Nevertheless, under these conditions IGF-I was not able to further increase Akt phosphorylation, reflecting the profound IGF-I resistance induced by the proinflammatory milieu. Altogether, our results suggest a negative cross-talk between signaling pathways triggered by proinflammatory cytokines and IGF-IR–mediated signaling in the retina at the level of IGF-IR and IRS1 that constitute a critical node of modulation of the insulin/IGF-I signaling network.12 This negative cross-talk was directly reflected by decreased Akt phosphorylation. Because it has been demonstrated that the neuroprotective action of IGF-I in vivo is most likely mediated by the PI 3-kinase/Akt pathway,59 our data strongly suggest that the proinflammatory milieu in the retina might compromise the fate of retinal cells. 
Protein tyrosine phosphatase 1B has emerged as a negative modulator of IR/IGF-IR tyrosine phosphorylation but most studies have focused on its role in inflammation linked to obesity and T2DM.39 In the retina, Rajala and coworkers60 demonstrated a negative modulation of IR tyrosine phosphorylation by PTP1B in diabetic mice and, conversely, enhanced IR phosphorylation in rod photoreceptors from PTP1B-deficient mice under light stress.25 As reported in fibroblasts,21 we have demonstrated that PTP1B modulates IGF-IR tyrosine phosphorylation in the retina.26 Thus, all these evidences prompted us to investigate the role of PTP1B in the retina under proinflammatory conditions. In 661W cells treated with proinflammatory cytokines both PTP1B mRNA protein levels and its enzymatic activity were elevated and this effect was also reflected in the whole retina. Interestingly, treatment of 661W cells with cytokines induced a cleavage of PTP1B similar to that reported in platelets that resulted in increased enzymatic activity.38 
Based on our previous work in which PTP1B deficiency enhanced the IGF-IR/Akt-mediated signaling in mouse retina,26 we used PTP1B siRNA to transiently decrease PTP1B in retinal cells and avoid constitutive adaptations of genetic deletion. This approach allowed us to maintain IGF-I responsiveness of 661W cells in the presence of proinflammatory cytokines as reflected by increased levels of IGF-IR and Akt phosphorylation compared with the scrambled condition. The pathophysiological relevance of these results was assessed in retinal explants in which we were able to reduce PTP1B levels by siRNA and recover IGF-IR/Akt sensitization in a proinflammatory environment as in the retinas from PTP1B−/− mice. We previously reported that PTP1B deficiency rescued IGF-IR tyrosine phosphorylation, but not Akt phosphorylation, in retinas from diabetic IRS2-deficient mice due to elevated PTEN levels.26 However, results presented herein indicate that PTP1B silencing maintained IGF-IR–mediated Akt phosphorylation in the presence of an inflammatory environment. An explanation for these differences could be the lack of effect of proinflammatory cytokines in modulating PTEN in retinal cells and explants (data not shown), suggesting the importance of this inositol phosphatase in blocking the pathway specifically upstream of Akt. 
Insulin/IGF-I resistance links obesity and/or T2DM to neurodegeneration, which has far-reaching effects in the CNS.6,61 In fact, both peripheral and central insulin resistance may be boosted by the chronic inflammatory environment associated with obesity. On the other hand, low serum IGF-I input to the brain may also exacerbate gliosis as IGF-I is involved in the inflammatory responses of glial cells.62 In this regard, our study has shown for the first time that a combination of proinflammatory cytokines is sufficient to induce reactive gliosis, monitored by levels of GAFP, in the retina and this effect is ameliorated by cotreatment with a PTP1B inhibitor, which might act by enhancing IGF-IR–mediated signaling as occurred in the insulin signaling.44 Although deleterious effects of high levels of IGF-I in the retina have been reported,6365 our previous data in retinal explants from mice with retinitis pigmentosa that presented hallmarks of inflammation, showed that treatment with IGF-I for 24 hours ameliorated reactive gliosis.66 A step further, PTP1B expression was elevated in the retina of diabetic db/db mice that showed systemic inflammation45 and reactive gliosis3 reinforcing the link between PTP1B and inflammation in the retina. Interestingly, reactive gliosis in retinal explants from db/db mice was reduced by treatment with a PTP1B inhibitor. As reactive gliosis is present in DR,3,6769 our results might have therapeutic implications proposing a novel strategy for its treatment based on the specific inhibition of PTP1B in the retina. 
In conclusion, our results show that retinal cells respond to the proinflammatory environment triggering signaling cascades that interfere with the IGF-IR/Akt survival pathway and induce reactive gliosis. These effects can be prevented by reducing PTP1B levels and strongly suggest that targeting this phosphatase might be useful for modulating the beneficial effects of IGF-I in the inflammatory states during DR. 
Acknowledgments
The authors thank the animal facility staff (IIB Alberto Sols, CSIC) for technical assistance, Lucía Sánchez-Ruiloba and Diego Navarro for assistance with microscopy, and Patricia Boya (CSIC, Spain) for critical reading of the manuscript. 
Supported by grants from Spanish Ministry of Economy and Competitivity (SAF2012-33283; Madrid, Spain), Comunidad de Madrid S2010/BMD-2423 (Madrid, Spain), Centro de Investigación Biomédica en Red de Diabetes y Enfermedades Metabólicas Asociadas (CIBEDEM, Instituto Carlos III, Madrid, Spain), and European Union (EUROCONDOR; FP7 HEALTH.2011.2.4.3.1.; Barcelona, Spain). 
Disclosure: A.I. Arroba, None; A.M. Valverde, None 
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Figure 1
 
Insulin-like growth factor-IR–mediated Akt signaling in 661W retinal cells. Confluent 661W cells were serum-starved for 2 hours and then stimulated with various doses of IGF-I for 15 minutes. Protein extracts (30 μg) were separated by SDS-PAGE and analyzed by Western blot with the anti-phospho IGF-IR (Tyr1135/1136), anti–IGF-IR, anti-phospho Akt (Ser473), anti-phospho Akt (Thr308), and anti-Akt antibodies. (A) Representative autoradiograms are shown (n = 3 independent experiments). (B) The blots were quantitated by scanning densitometry. Levels of phosphorylated IGF-IR and Akt were normalized by their corresponding levels of total IGF-I or Akt protein, respectively, that were used as loading control. Results are expressed as fold increase of IGF-I stimulation and are means ± SEM. Data were analyzed by 1-way ANOVA followed by Bonferroni t-test. Comparisons were *†‡P ≤ 0.05 IGF-I stimulation versus their corresponding nonstimulated basal (B).
Figure 1
 
Insulin-like growth factor-IR–mediated Akt signaling in 661W retinal cells. Confluent 661W cells were serum-starved for 2 hours and then stimulated with various doses of IGF-I for 15 minutes. Protein extracts (30 μg) were separated by SDS-PAGE and analyzed by Western blot with the anti-phospho IGF-IR (Tyr1135/1136), anti–IGF-IR, anti-phospho Akt (Ser473), anti-phospho Akt (Thr308), and anti-Akt antibodies. (A) Representative autoradiograms are shown (n = 3 independent experiments). (B) The blots were quantitated by scanning densitometry. Levels of phosphorylated IGF-IR and Akt were normalized by their corresponding levels of total IGF-I or Akt protein, respectively, that were used as loading control. Results are expressed as fold increase of IGF-I stimulation and are means ± SEM. Data were analyzed by 1-way ANOVA followed by Bonferroni t-test. Comparisons were *†‡P ≤ 0.05 IGF-I stimulation versus their corresponding nonstimulated basal (B).
Figure 2
 
A combination of proinflammatory cytokines (CK) induced the activation of stress kinases and the phosphorylation of IRS1 at serine 307 in retinal cells. (A) 661W photoreceptors and (C) mouse retinal explants were stimulated with a combination of proinflammatory cytokines containing TNFα, IL6, and IL1β (CK; 20 ng/mL each) at the indicated time-periods. Protein extracts (50 μg) were separated by SDS-PAGE and the phosphorylation of IRS1 (Ser307), JNK (Thr183/Tyr185), and p38 MAPK (Thr180/Tyr182) was analyzed by Western blot with the corresponding antibodies. Total levels of IRS1, JNK, and p38 MAPK were also analyzed. Representative autoradiograms are shown (n = 3 independent experiments). In (B) and (D) the results are means ± SEM. Data were analyzed by 1-way ANOVA followed by Bonferroni t-test. Comparisons were *†‡P ≤ 0.05 cytokines (CK) versus their corresponding nonstimulated basal (B).
Figure 2
 
A combination of proinflammatory cytokines (CK) induced the activation of stress kinases and the phosphorylation of IRS1 at serine 307 in retinal cells. (A) 661W photoreceptors and (C) mouse retinal explants were stimulated with a combination of proinflammatory cytokines containing TNFα, IL6, and IL1β (CK; 20 ng/mL each) at the indicated time-periods. Protein extracts (50 μg) were separated by SDS-PAGE and the phosphorylation of IRS1 (Ser307), JNK (Thr183/Tyr185), and p38 MAPK (Thr180/Tyr182) was analyzed by Western blot with the corresponding antibodies. Total levels of IRS1, JNK, and p38 MAPK were also analyzed. Representative autoradiograms are shown (n = 3 independent experiments). In (B) and (D) the results are means ± SEM. Data were analyzed by 1-way ANOVA followed by Bonferroni t-test. Comparisons were *†‡P ≤ 0.05 cytokines (CK) versus their corresponding nonstimulated basal (B).
Figure 3
 
Proinflammatory cytokines activated signaling pathways that negatively modulated IGF-IR–mediated signaling. 661W cells (AC) and retinal explants (D, E) were treated with cytokines (CK; 20 ng/mL) for 24 hours, serum-starved for 2 hours, and further stimulated with IGF-I (I; 10 nM) for 15 minutes (661W retinal cells) or IGF-I (I; 20 nM) for 30 minutes (retinal explants). In (B) (661W cells) and (E) (retinal explants) the treatment was 24 hours with cytokines. Protein extracts (50 μg) were separated by SDS-PAGE and analyzed by Western blot with antibodies against phospho–IGF-IR (Tyr1135/1366), IGF-IR, phospho-Akt (Ser473), phospho-Akt (Thr308), Akt, IRS1, and α-tubulin. Representative autoradiograms are shown (n = 6 independent experiments). The blots were quantitated by scanning densitometry. Levels of phosphorylated proteins (pIGF-IR and pAkt) were normalized by levels of their corresponding total IGF-IR and Akt protein that were used as loading controls. These results are expressed as fold change of IGF-I stimulation related to each basal. Alpha-tubulin was used as a loading control for IRS1. The results are means ± SEM. Data were analyzed by 1- or 2-way ANOVA followed by Bonferroni t-test. In (A, C, D) comparisons were *P ≤ 0.05 IGF-I (I) versus nonstimulated basal (B); †P ≤ 0.05 cytokines plus IGF-I (CKI) versus IGF-I (I). In (B) and (E) results are means ± SEM. Data were analyzed by paired t-test. Comparisons were *P ≤ 0.05 cytokines (CK) versus nonstimulated basal (B).
Figure 3
 
Proinflammatory cytokines activated signaling pathways that negatively modulated IGF-IR–mediated signaling. 661W cells (AC) and retinal explants (D, E) were treated with cytokines (CK; 20 ng/mL) for 24 hours, serum-starved for 2 hours, and further stimulated with IGF-I (I; 10 nM) for 15 minutes (661W retinal cells) or IGF-I (I; 20 nM) for 30 minutes (retinal explants). In (B) (661W cells) and (E) (retinal explants) the treatment was 24 hours with cytokines. Protein extracts (50 μg) were separated by SDS-PAGE and analyzed by Western blot with antibodies against phospho–IGF-IR (Tyr1135/1366), IGF-IR, phospho-Akt (Ser473), phospho-Akt (Thr308), Akt, IRS1, and α-tubulin. Representative autoradiograms are shown (n = 6 independent experiments). The blots were quantitated by scanning densitometry. Levels of phosphorylated proteins (pIGF-IR and pAkt) were normalized by levels of their corresponding total IGF-IR and Akt protein that were used as loading controls. These results are expressed as fold change of IGF-I stimulation related to each basal. Alpha-tubulin was used as a loading control for IRS1. The results are means ± SEM. Data were analyzed by 1- or 2-way ANOVA followed by Bonferroni t-test. In (A, C, D) comparisons were *P ≤ 0.05 IGF-I (I) versus nonstimulated basal (B); †P ≤ 0.05 cytokines plus IGF-I (CKI) versus IGF-I (I). In (B) and (E) results are means ± SEM. Data were analyzed by paired t-test. Comparisons were *P ≤ 0.05 cytokines (CK) versus nonstimulated basal (B).
Figure 4
 
Protein tyrosine phosphatase 1B expression is upregulated in retinal cells and retinal explants cultured under proinflammatory conditions. (AC) For 24 hours, 661W cells were treated with cytokines (CK; 20 ng/mL). (A) Protein tyrosine phosphatase 1B mRNA levels were determined by real-time PCR. Used as a control was 18S RNA. Results are expressed as 2−ΔCt. (B) Protein extracts (50 μg) were separated by SDS-PAGE and analyzed by Western blot with the anti-PTP1B antibody (Ref. 05-1087; Millipore). Alpha-tubulin was used as a loading control. Representative autoradiograms are shown. (C) Protein tyrosine phosphatase 1B enzymatic activity was analyzed as described in Materials and Methods. The results of PTP1B enzymatic activity are expressed as picomoles of released phosphate/min/g protein. The results are means ± SEM (n = 5 independent experiments). Data were analyzed by Student's paired t-test; *P ≤ 0.05 cytokines (CK) versus nonstimulated basal (B). Retinal explants were treated with cytokines (CK; 20 ng/mL) for 24 hours. (D) Protein tyrosine phosphatase 1B mRNA levels were determined by real-time PCR. Used as a control was 18S RNA. Results are expressed as 2−ΔCt. (E) Protein extracts (50 μg) were separated by SDS-PAGE and analyzed by Western blot with the anti-PTP1B antibody (Ref. 05-1087; Millipore). Alpha-tubulin was used as a loading control. The results are expressed as fold change and are means ± SEM (n = 12 independent retinas per condition). Data were analyzed by Student's paired t-test. Comparisons were *P ≤ 0.05 cytokines (CK) versus nonstimulated basal (B).
Figure 4
 
Protein tyrosine phosphatase 1B expression is upregulated in retinal cells and retinal explants cultured under proinflammatory conditions. (AC) For 24 hours, 661W cells were treated with cytokines (CK; 20 ng/mL). (A) Protein tyrosine phosphatase 1B mRNA levels were determined by real-time PCR. Used as a control was 18S RNA. Results are expressed as 2−ΔCt. (B) Protein extracts (50 μg) were separated by SDS-PAGE and analyzed by Western blot with the anti-PTP1B antibody (Ref. 05-1087; Millipore). Alpha-tubulin was used as a loading control. Representative autoradiograms are shown. (C) Protein tyrosine phosphatase 1B enzymatic activity was analyzed as described in Materials and Methods. The results of PTP1B enzymatic activity are expressed as picomoles of released phosphate/min/g protein. The results are means ± SEM (n = 5 independent experiments). Data were analyzed by Student's paired t-test; *P ≤ 0.05 cytokines (CK) versus nonstimulated basal (B). Retinal explants were treated with cytokines (CK; 20 ng/mL) for 24 hours. (D) Protein tyrosine phosphatase 1B mRNA levels were determined by real-time PCR. Used as a control was 18S RNA. Results are expressed as 2−ΔCt. (E) Protein extracts (50 μg) were separated by SDS-PAGE and analyzed by Western blot with the anti-PTP1B antibody (Ref. 05-1087; Millipore). Alpha-tubulin was used as a loading control. The results are expressed as fold change and are means ± SEM (n = 12 independent retinas per condition). Data were analyzed by Student's paired t-test. Comparisons were *P ≤ 0.05 cytokines (CK) versus nonstimulated basal (B).
Figure 5
 
Reduction of PTP1B levels by siRNA in retinal cells protected against inflammation-mediated inhibition of IGF-IR signaling. (A) Efficiency of PTP1B siRNA in decreasing PTP1B levels in 661W cells. (B, C) For 36 hours, 661W cells were transfected with scrambled or PTP1B siRNA oligonucleotides and transfected cells were treated with cytokines (CK; 20 ng/mL) for 24 hours, serum-starved for 2 hours and further stimulated with IGF-I (10 nM) for 15 minutes. (D) Efficiency of PTP1B siRNA in decreasing PTP1B in retinal explants. (E, F) Retinal explants were treated with siRNAs and stimulated as described for 661W cells. (G) Retinal explants from PTP1B−/− mice were stimulated with cytokines and IGF-I as indicated above. Protein extracts (50 μg) were separated by SDS-PAGE and analyzed by Western blot with the corresponding antibodies: anti-PTP1B (Ref. 07-088; Millipore), anti-phospho IGF-IR (Tyr 1135/1136), anti-IGF-IR, anti-phospho Akt (Ser473), anti-Akt, and anti–α-tubulin. Representative autoradiograms are shown (n = 5 independent experiments or retinas per experimental condition). Autoradiograms were quantitated by scanning densitometry. Levels of phosphorylated proteins (pIGF-IR and pAkt) were normalized by levels of corresponding total (IGF-IR and Akt) proteins that were used as loading controls. These results are expressed as fold change of IGF-I stimulation related to each basal. Levels of PTP1B were normalized by α-tubulin. The results are means ± SEM. In (A) and (D), data were analyzed by paired t-test and comparisons were *P ≤ 0.05 siRNA PTP1B versus scrambled siRNA (SC). In (C) and (F), data were analyzed by 1- or 2-way ANOVA followed by Bonferroni t-test. Comparisons were *P ≤ 0.05 CKI siRNA PTP1B versus CKI SC.
Figure 5
 
Reduction of PTP1B levels by siRNA in retinal cells protected against inflammation-mediated inhibition of IGF-IR signaling. (A) Efficiency of PTP1B siRNA in decreasing PTP1B levels in 661W cells. (B, C) For 36 hours, 661W cells were transfected with scrambled or PTP1B siRNA oligonucleotides and transfected cells were treated with cytokines (CK; 20 ng/mL) for 24 hours, serum-starved for 2 hours and further stimulated with IGF-I (10 nM) for 15 minutes. (D) Efficiency of PTP1B siRNA in decreasing PTP1B in retinal explants. (E, F) Retinal explants were treated with siRNAs and stimulated as described for 661W cells. (G) Retinal explants from PTP1B−/− mice were stimulated with cytokines and IGF-I as indicated above. Protein extracts (50 μg) were separated by SDS-PAGE and analyzed by Western blot with the corresponding antibodies: anti-PTP1B (Ref. 07-088; Millipore), anti-phospho IGF-IR (Tyr 1135/1136), anti-IGF-IR, anti-phospho Akt (Ser473), anti-Akt, and anti–α-tubulin. Representative autoradiograms are shown (n = 5 independent experiments or retinas per experimental condition). Autoradiograms were quantitated by scanning densitometry. Levels of phosphorylated proteins (pIGF-IR and pAkt) were normalized by levels of corresponding total (IGF-IR and Akt) proteins that were used as loading controls. These results are expressed as fold change of IGF-I stimulation related to each basal. Levels of PTP1B were normalized by α-tubulin. The results are means ± SEM. In (A) and (D), data were analyzed by paired t-test and comparisons were *P ≤ 0.05 siRNA PTP1B versus scrambled siRNA (SC). In (C) and (F), data were analyzed by 1- or 2-way ANOVA followed by Bonferroni t-test. Comparisons were *P ≤ 0.05 CKI siRNA PTP1B versus CKI SC.
Figure 6
 
Pharmacologic inhibition of PTP1B reduced reactive gliosis induced by proinflammatory cytokines in retinal explants. Whole retinas were obtained from 10-week-old mice and cultured with cytokines (CK; 20 ng/mL) for 24 hours in the presence or absence of the PTP1B inhibitor (10 μM). (A) Protein extracts (50 μg) were separated by SDS-PAGE and analyzed by Western blot with the corresponding antibodies against GFAP and α-tubulin that was used as a loading control. Representative autoradiograms are shown. Autoradiograms corresponding to GFAP levels were quantitated by scanning densitometry. The results are means ± SEM (n = 5 retinas per experimental condition). Data were analyzed by 1-way ANOVA followed by Bonferroni t-test. Comparisons were *P ≤ 0.05 cytokines (CK) versus basal (B); †P ≤ 0.05 CK plus PTP1B inhibitor versus CK. (B) Whole retinas were obtained from 10-week-old mice, cultured as described in (A) an immunostaining for GFAP (green) was carried out. Five retinas per experimental condition were analyzed. Representative confocal images are shown. Quantification of immunofluorescence was performed as described in Methods. Data were analyzed by paired t-test. Comparisons were *P ≤ 0.05 CK versus basal (B).
Figure 6
 
Pharmacologic inhibition of PTP1B reduced reactive gliosis induced by proinflammatory cytokines in retinal explants. Whole retinas were obtained from 10-week-old mice and cultured with cytokines (CK; 20 ng/mL) for 24 hours in the presence or absence of the PTP1B inhibitor (10 μM). (A) Protein extracts (50 μg) were separated by SDS-PAGE and analyzed by Western blot with the corresponding antibodies against GFAP and α-tubulin that was used as a loading control. Representative autoradiograms are shown. Autoradiograms corresponding to GFAP levels were quantitated by scanning densitometry. The results are means ± SEM (n = 5 retinas per experimental condition). Data were analyzed by 1-way ANOVA followed by Bonferroni t-test. Comparisons were *P ≤ 0.05 cytokines (CK) versus basal (B); †P ≤ 0.05 CK plus PTP1B inhibitor versus CK. (B) Whole retinas were obtained from 10-week-old mice, cultured as described in (A) an immunostaining for GFAP (green) was carried out. Five retinas per experimental condition were analyzed. Representative confocal images are shown. Quantification of immunofluorescence was performed as described in Methods. Data were analyzed by paired t-test. Comparisons were *P ≤ 0.05 CK versus basal (B).
Figure 7
 
Pharmacologic inhibition of PTP1B reduced reactive gliosis in db/db mice. Whole retinas were obtained from 10-week-old db/+ and db/db mice. (A) Protein tyrosine phosphatase 1B mRNA levels were determined by real-time PCR. As a control, 18S RNA was used. Results are expressed as 2−ΔCt. (B) Protein extracts (50 μg) were separated by SDS-PAGE and analyzed by Western blot with the anti-PTP1B antibody (Ref. 05-1087; Millipore). Alpha-tubulin was used as a loading control. Representative autoradiograms are shown. The results are means ± SEM (n = 12 independent retinas per condition). Data were analyzed by Student's paired t-test. Comparisons were *P ≤ 0.05 db/db versus db/+. (C) Immunostaining for GFAP (green) was carried out in retinas from db/+ and db/db mice (upper panels) and in retinas from db/db mice were cultured for 24 hours in the presence or absence of the PTP1B inhibitor (10 μM; lower panels). Five retinas per experimental condition were analyzed. Representative images are shown. Quantification of immunofluorescence was performed as described in Methods. Data were analyzed by paired t-test. Comparisons were *P ≤ 0.05 db/db versus db/+ and †P ≤ 0.05 db/db PTP1B inhibitor versus db/db basal.
Figure 7
 
Pharmacologic inhibition of PTP1B reduced reactive gliosis in db/db mice. Whole retinas were obtained from 10-week-old db/+ and db/db mice. (A) Protein tyrosine phosphatase 1B mRNA levels were determined by real-time PCR. As a control, 18S RNA was used. Results are expressed as 2−ΔCt. (B) Protein extracts (50 μg) were separated by SDS-PAGE and analyzed by Western blot with the anti-PTP1B antibody (Ref. 05-1087; Millipore). Alpha-tubulin was used as a loading control. Representative autoradiograms are shown. The results are means ± SEM (n = 12 independent retinas per condition). Data were analyzed by Student's paired t-test. Comparisons were *P ≤ 0.05 db/db versus db/+. (C) Immunostaining for GFAP (green) was carried out in retinas from db/+ and db/db mice (upper panels) and in retinas from db/db mice were cultured for 24 hours in the presence or absence of the PTP1B inhibitor (10 μM; lower panels). Five retinas per experimental condition were analyzed. Representative images are shown. Quantification of immunofluorescence was performed as described in Methods. Data were analyzed by paired t-test. Comparisons were *P ≤ 0.05 db/db versus db/+ and †P ≤ 0.05 db/db PTP1B inhibitor versus db/db basal.
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