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• W2004608929 abstract "High glucose (30 mm) and high insulin (1 nm), pathogenic factors of type 2 diabetes, increased mRNA expression and synthesis of lamininβ1 and fibronectin after 24 h of incubation in kidney proximal tubular epithelial (MCT) cells. We tested the hypothesis that inactivation of glycogen synthase kinase 3β (GSK3β) by high glucose and high insulin induces increase in synthesis of laminin β1 via activation of eIF2Bϵ. Both high glucose and high insulin induced Ser-9 phosphorylation and inactivation of GSK3β at 2 h that lasted for up to 48 h. This was associated with dephosphorylation of eIF2Bϵ and eEF2, and increase in phosphorylation of 4E-BP1 and eIF4E. Expression of the kinase-dead mutant of GSK3β or constitutively active kinase led to increased and diminished laminin β1 synthesis, respectively. Incubation with selective kinase inhibitors showed that high glucose- and high insulin-induced laminin β1 synthesis and phosphorylation of GSK3β were dependent on PI 3-kinase, Erk, and mTOR. High glucose and high insulin augmented activation of Akt, Erk, and p70S6 kinase. Dominant negative Akt, but not dominant negative p70S6 kinase, inhibited GSK3β phosphorylation induced by high glucose and high insulin, suggesting Akt but not p70S6 kinase was upstream of GSK3β. Status of GSK3β was examined in vivo in renal cortex of db/db mice with type 2 diabetes at 2 weeks and 2 months of diabetes. Diabetic mice showed increased phosphorylation of renal cortical GSK3β and decreased phosphorylation of eIF2Bϵ, which correlated with renal hypertrophy at 2 weeks, and increased laminin β1 and fibronectin protein content at 2 months. GSK3β and eIF2Bϵ play a role in augmented protein synthesis associated with high glucose- and high insulin-stimulated hypertrophy and matrix accumulation in renal disease in type 2 diabetes. High glucose (30 mm) and high insulin (1 nm), pathogenic factors of type 2 diabetes, increased mRNA expression and synthesis of lamininβ1 and fibronectin after 24 h of incubation in kidney proximal tubular epithelial (MCT) cells. We tested the hypothesis that inactivation of glycogen synthase kinase 3β (GSK3β) by high glucose and high insulin induces increase in synthesis of laminin β1 via activation of eIF2Bϵ. Both high glucose and high insulin induced Ser-9 phosphorylation and inactivation of GSK3β at 2 h that lasted for up to 48 h. This was associated with dephosphorylation of eIF2Bϵ and eEF2, and increase in phosphorylation of 4E-BP1 and eIF4E. Expression of the kinase-dead mutant of GSK3β or constitutively active kinase led to increased and diminished laminin β1 synthesis, respectively. Incubation with selective kinase inhibitors showed that high glucose- and high insulin-induced laminin β1 synthesis and phosphorylation of GSK3β were dependent on PI 3-kinase, Erk, and mTOR. High glucose and high insulin augmented activation of Akt, Erk, and p70S6 kinase. Dominant negative Akt, but not dominant negative p70S6 kinase, inhibited GSK3β phosphorylation induced by high glucose and high insulin, suggesting Akt but not p70S6 kinase was upstream of GSK3β. Status of GSK3β was examined in vivo in renal cortex of db/db mice with type 2 diabetes at 2 weeks and 2 months of diabetes. Diabetic mice showed increased phosphorylation of renal cortical GSK3β and decreased phosphorylation of eIF2Bϵ, which correlated with renal hypertrophy at 2 weeks, and increased laminin β1 and fibronectin protein content at 2 months. GSK3β and eIF2Bϵ play a role in augmented protein synthesis associated with high glucose- and high insulin-stimulated hypertrophy and matrix accumulation in renal disease in type 2 diabetes. Glycogen synthase kinase 3β (GSK3β) 3The abbreviations used are: GSK, glycogen synthase kinase; MOPS, 4-morpholinepropanesulfonic acid; ANOVA, analysis of variance; PI, phosphatidylinositol; Erk, extracellular signal-regulated kinase; MAP, mitogen-activated protein; HA, hemagglutinin; eIF, eukaryotic translation initiation factor. 3The abbreviations used are: GSK, glycogen synthase kinase; MOPS, 4-morpholinepropanesulfonic acid; ANOVA, analysis of variance; PI, phosphatidylinositol; Erk, extracellular signal-regulated kinase; MAP, mitogen-activated protein; HA, hemagglutinin; eIF, eukaryotic translation initiation factor. was originally identified as an enzyme required for the regulation of glycogen metabolism (1Embi N. Rylatt D.B. Cohen P. Eur. J. Biochem. 1980; 107: 519-527Crossref PubMed Scopus (807) Google Scholar). However, it has been found to be involved in a variety of cellular responses including cytoskeletal regulation (2Hanger D.P. Hughes K. Woodgett J.R. Brion J.P. Anderton B.H. Neurosci. Lett. 1992; 147: 58-62Crossref PubMed Scopus (653) Google Scholar), cell cycle progression (3Cui H. Meng Y. Bulleit R.F. Brain Res. Dev. Brain Res. 1998; 111: 177-188Crossref PubMed Scopus (75) Google Scholar, 4Diehl J.A. Cheng M. Roussel M.F. Sherr C.J. Genes Dev. 1998; 12: 3499-3511Crossref PubMed Scopus (1858) Google Scholar), apoptosis (5Beurel E. Kornprobst M. Blivet-Van Eggelpoel M.J. Ruiz-Ruiz C. Cadoret A. Capeau J. Desbois-Mouthon C. Exp. Cell Res. 2004; 300: 354-364Crossref PubMed Scopus (72) Google Scholar, 6Linseman D.A. Butts B.D. Precht T.A. Phelps R.A. Le S.S. Laessig T.A. Bouchard R.J. Florez-McClure M.L. Heidenreich K.A. J. Neurosci. 2004; 24: 9993-10002Crossref PubMed Scopus (324) Google Scholar), and cell adhesion (7Cordes N. van Beuningen D. Br. J. Cancer. 2003; 88: 1470-1479Crossref PubMed Scopus (72) Google Scholar). GSK3β regulation of protein synthesis has not been completely understood. GSK3β acts as a switch that regulates both transcription and mRNA translation by controlling the activity of transcription factors and eukaryotic translation initiation factor 2Bϵ (eIF2Bϵ), respectively. In the resting cell, GSK3β is unphosphorylated and active, inhibiting the activity of its substrates, e.g. eIF2Bϵ and glycogen synthase. Upon stimulation, GSK3β is phosphorylated on Ser-9 and inactivated, leading to release of inhibition on activity of its substrates. Several kinases are implicated in Ser-9 phosphorylation of GSK3β including p70S6 kinase (8Cross D.A. Alessi D.R. Cohen P. Andjelkovich M. Hemmings B.A. Nature. 1995; 378: 785-789Crossref PubMed Scopus (4364) Google Scholar), p90RSK (9Eldar-Finkelman H. Seger R. Vandenheede J.R. Krebs E.G. J. Biol. Chem. 1995; 270: 987-990Abstract Full Text Full Text PDF PubMed Scopus (192) Google Scholar), PKC, PKA (10De Servi B. Hermani A. Medunjanin S. Mayer D. Oncogene. 2005; 24: 4946-4955Crossref PubMed Scopus (35) Google Scholar, 11Hagen T. Cross D.A. Culbert A.A. West A. Frame S. Morrice N. Reith A.D. J. Biol. Chem. 2006; 281: 35021-35029Abstract Full Text Full Text PDF PubMed Scopus (15) Google Scholar, 12Ossipova O. Bardeesy N. DePinho R.A. Green J.B. Nat. Cell Biol. 2003; 5: 889-894Crossref PubMed Scopus (115) Google Scholar), and Akt (13Altomare D.A. Wang H.Q. Skele K.L. De Rienzo A. Klein-Szanto A.J. Godwin A.K. Testa J.R. Oncogene. 2004; 23: 5853-5857Crossref PubMed Scopus (349) Google Scholar, 14Trivedi C.M. Luo Y. Yin Z. Zhang M. Zhu W. Wang T. Floss T. Goettlicher M. Noppinger P.R. Wurst W. Ferrari V.A. Abrams C.S. Gruber P.J. Epstein J.A. Nat. Med. 2007; 13: 324-331Crossref PubMed Scopus (379) Google Scholar). Augmented protein synthesis contributes to two cardinal manifestations of diabetic kidney disease, i.e. renal hypertrophy and accumulation of extracellular matrix proteins (15Kasinath B.S. Lee M.J. Feliers D. Sonenberg N. Cortes P.M.C. Contemporary Diabetes. The Diabetic Kidney. Humana Press, NJ2003: 97-116Google Scholar). However, the role of GSK3β in these events has not been investigated. Signaling mechanisms favoring renal hypertrophy have received much attention (16Chen J. Chen J.K. Neilson E.G. Harris R.C. J. Am. Soc. Nephrol. 2006; 17: 1615-1623Crossref PubMed Scopus (52) Google Scholar, 17Lee M.J. Feliers D. Mariappan M.M. Sataranatarajan K. Mahimainathan L. Musi N. Foretz M. Viollet B. Weinberg J.M. Choudhury G.G. Kasinath B.S. Am. J. Physiol. Renal Physiol. 2007; 292: F617-F627Crossref PubMed Scopus (246) Google Scholar, 18Lloberas N. Cruzado J.M. Franquesa M. Herrero-Fresneda I. Torras J. Alperovich G. Rama I. Vidal A. Grinyo J.M. J. Am. Soc. Nephrol. 2006; 17: 1395-1404Crossref PubMed Scopus (223) Google Scholar, 19Mariappan M.M. Senthil D. Natarajan K.S. Choudhury G.G. Kasinath B.S. J. Biol. Chem. 2005; 280: 28402-28411Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 20Sakaguchi M. Isono M. Isshiki K. Sugimoto T. Koya D. Kashiwagi A. Biochem. Biophys. Res. Commun. 2006; 340: 296-301Crossref PubMed Scopus (144) Google Scholar, 21Sataranatarajan K. Mariappan M.M. Lee M.J. Feliers D. Choudhury G.G. Barnes J.L. Kasinath B.S. Am. J. Pathol. 2007; 171: 1733-1742Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 22Senthil D. Choudhury G.G. Abboud H.E. Sonenberg N. Kasinath B.S. Am. J. Physiol. Renal Physiol. 2002; 283: F1226-F1236Crossref PubMed Scopus (38) Google Scholar, 23Senthil D. Choudhury G.G. McLaurin C. Kasinath B.S. Kidney Int. 2003; 64: 468-479Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar, 24Senthil D. Faulkner J.L. Choudhury G.G. Abboud H.E. Kasinath B.S. Biochem. J. 2001; 360: 87-95Crossref PubMed Scopus (27) Google Scholar, 25Senthil D. Ghosh Choudhury G. Bhandari B.K. Kasinath B.S. Biochem. J. 2002; 368: 49-56Crossref PubMed Google Scholar). In contrast, constitutive signaling mechanisms that counteract the prohypertrophic signaling mechanisms and check unrestricted progression of protein synthesis are not well understood. GSK3β appears to constitutively inhibit protein synthesis by its inhibition of eIF2Bϵ. We hypothesized that inactivation of GSK3β by high glucose and high insulin induces an increase in synthesis of laminin β1 via activation of eIF2Bϵ. We investigated if GSK3β is inactive in the kidney in type 2 diabetes. We employed both in vitro cell culture and in vivo animal models of type 2 diabetes in our investigation. Cell Culture—SV-40-immortalized murine kidney proximal tubular epithelial (MCT) cells (kindly provided by Dr. Eric Neilson, Vanderbilt University) were grown in Dulbecco's modified Eagle's medium containing 7% fetal bovine serum, 5 mm glucose, 100 units/ml penicillin, 100 μg/ml streptomycin, and 2 mm glutamine. MCT cells express in vivo properties of proximal tubular epithelial cells (26Haverty T.P. Kelly C.J. Hines W.H. Amenta P.S. Watanabe M. Harper R.A. Kefalides N.A. Neilson E.G. J. Cell Biol. 1988; 107: 1359-1368Crossref PubMed Scopus (276) Google Scholar). Confluent cells were growth-arrested for 18 h in serum-free Dulbecco's modified Eagle's medium before experiment (23Senthil D. Choudhury G.G. McLaurin C. Kasinath B.S. Kidney Int. 2003; 64: 468-479Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar). Immunoblotting—Equal amounts of protein from cells were separated by SDS-PAGE, transferred to a nitrocellulose membrane, and probed with primary antibody for 3 h (19Mariappan M.M. Senthil D. Natarajan K.S. Choudhury G.G. Kasinath B.S. J. Biol. Chem. 2005; 280: 28402-28411Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar, 27Mariappan M.M. Feliers D. Mummidi S. Choudhury G.G. Kasinath B.S. Diabetes. 2007; 56: 476-485Crossref PubMed Scopus (67) Google Scholar). Primary antibodies were from Cell Signaling (Beverly, MA) if not otherwise mentioned. Laminin β1 and GSK3β antibody were from Santa Cruz Biotechnology, fibronectin antibody was from Sigma, and phospho-eIF2Bϵ (Ser-539) was purchased from Upstate, Lake Placid, NY. After washing, the membrane was incubated with peroxidase-conjugated secondary antibody (Jackson ImmunoResearch Laboratories Inc, West Grove, PA). Proteins were visualized by chemiluminescence using the ECL reagent (Pierce Biotechnology, Rockford, IL). Images of the bands were scanned by reflectance scanning densitometry, and the intensity of the bands was quantified using Scion Image software (27Mariappan M.M. Feliers D. Mummidi S. Choudhury G.G. Kasinath B.S. Diabetes. 2007; 56: 476-485Crossref PubMed Scopus (67) Google Scholar). For immunoblot analysis of 4E-BP1 phosphorylation, cell lysates were heated at 100 °C for 7 min, and treated with 15% trichloroacetic acid. Immunoblotting was performed using an antibody specific for 4E-BP1 phosphorylated at Thr-37/46 (27Mariappan M.M. Feliers D. Mummidi S. Choudhury G.G. Kasinath B.S. Diabetes. 2007; 56: 476-485Crossref PubMed Scopus (67) Google Scholar). GSK3β Immunokinase Activity Assay—Following treatment with high glucose or high insulin, cell lysates were prepared using the lysis buffer consisting of 50 mm Tris, 150 mm NaCl, 10% (v/v) glycerol, 0.5% (v/v) Nonidet P-40, 1 mm EDTA, 1 mm dithiothreitol, 500 μm Na3VO4, 500 μm NaF, 100 μm β-glycerophosphate, 100 μm sodium pyrophosphate, 10 μg/ml leupeptin, and 1% (v/v) aprotinin. 300 μg of total protein was incubated with 1 μg of monoclonal anti GSK3β (sc-81462) in a rotor for 12 h at 4 °C. The immune complexes were isolated by the addition of 25 μl of a slurry of protein A/G-agarose (sc-2003) and incubation for 2 h at 4 °C. Immunoprecipitates were washed twice with lysis buffer and twice with kinase reaction buffer (8 mm MOPS, pH 7.4, 0.2 mm EDTA, 10 mm magnesium acetate, 1 mm Na3VO4, 1 mm dithiothreitol, 2.5 mm β-glycerophosphate, 10 μg/ml leupeptin, and 1% (v/v) aprotinin). The kinase activity assays were performed in 40 μl of total reaction buffer containing 62.5 μm GSK3β substrate (BioMol, Plymouth, PA), 20 mm MgCl2, 125 μm ATP, and 10 μCi [γ-32P]ATP. The reaction mixture was allowed to proceed for 30 min at 30 °C, and 15 μl of the supernatant were spotted onto Whatman P81 phosphocellulose paper. The filter squares were washed five times for 5 min each in 0.75% phosphoric acid. The filters were then briefly rinsed in acetone, dried at room temperature, and subjected to liquid scintillation counting (28van der Velden J.L. Langen R.C. Kelders M.C. Wouters E.F. Janssen-Heininger Y.M. Schols A.M. Am. J. Physiol. Cell Physiol. 2006; 290: C453-C462Crossref PubMed Scopus (85) Google Scholar). Real Time RT-PCR—MCT cells were lysed, and their total RNA was isolated by acid phenol extraction using TRIzol (Invitrogen, Carlsbad, CA). Equal amounts of total RNA (1 μg) from MCT cells were converted to first-strand cDNA by using reverse transcriptase. PCR primer sequences for amplification of mouse laminin β1 (Primer Bank ID 21595540a2), mouse fibronectin (Primer Bank ID 4218966a1) and mouse GAPDH (Primer Bank ID 6679937a1) were obtained from Primer Bank, a public resource for PCR primers (29Wang X. Seed B. Nucleic Acids Res. 2003; 31: e154Crossref PubMed Scopus (680) Google Scholar). 2 μl of cDNA was amplified using SYBR Green PCR Master mix (Applied Biosystems, Foster City, CA) containing 100 nm forward and reverse primers. PCR amplification was performed using 7900HT Sequence Detection System (Applied Biosystems) using the manufacturer's protocol as recently described (21Sataranatarajan K. Mariappan M.M. Lee M.J. Feliers D. Choudhury G.G. Barnes J.L. Kasinath B.S. Am. J. Pathol. 2007; 171: 1733-1742Abstract Full Text Full Text PDF PubMed Scopus (109) Google Scholar, 27Mariappan M.M. Feliers D. Mummidi S. Choudhury G.G. Kasinath B.S. Diabetes. 2007; 56: 476-485Crossref PubMed Scopus (67) Google Scholar). Dissociation curve analysis was performed following PCR amplification to confirm the specificity of the primers. Relative mRNA expression was calculated using the ΔΔCt method. Transfection Studies—MCT cells were transiently transfected with plasmids containing the hemagglutinin (HA)-tagged kinase-dead (K85A) and constitutively active (S9A) constructs for GSK3β (Plasmids 14755 and 14754, respectively, Addgene, Cambridge, MA, deposited by Dr. Jim Woodgett) (30Stambolic V. Woodgett J.R. Biochem. J. 1994; 303: 701-704Crossref PubMed Scopus (506) Google Scholar) using Lipofectamine-Plus reagent (Invitrogen) (19Mariappan M.M. Senthil D. Natarajan K.S. Choudhury G.G. Kasinath B.S. J. Biol. Chem. 2005; 280: 28402-28411Abstract Full Text Full Text PDF PubMed Scopus (25) Google Scholar). Plasmid containing kinase-dead p70S6 kinase was also purchased from Addgene (Plasmid 8985, deposited by Dr. John Blenis) (31Schalm S.S. Blenis J. Curr. Biol. 2002; 12: 632-639Abstract Full Text Full Text PDF PubMed Scopus (384) Google Scholar). Infection of MCT cells was carried out with (100 moi) of adenoviral vector expressing dominant negative HA-tagged Akt (Ad-DN-Akt) (32Choudhury G.G. J. Biol. Chem. 2001; 276: 35636-35643Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar), and adenovirus containing green fluorescence protein (Ad-GFP) was used as the control. Animal Study—The C57BL6/KsJ lepr-/-db/db mice, a model of type 2 diabetes, and its lean littermates (db/m) (Jackson Laboratory, Bar Harbor, ME), were maintained on regular laboratory chow. Blood glucose concentration was monitored for emergence of diabetes. In the present study, lean littermate control and diabetic mice were studied in the early phase, after 2 weeks of onset of hyperglycemia, and, in the late phase, at 2 months of hyperglycemia. Previously we have reported that db/db mice display renal hypertrophy at 2 weeks (23Senthil D. Choudhury G.G. McLaurin C. Kasinath B.S. Kidney Int. 2003; 64: 468-479Abstract Full Text Full Text PDF PubMed Scopus (77) Google Scholar) and increase in laminin content at 2 months of diabetes (33Ha T.S. Barnes J.L. Stewart J.L. Ko C.W. Miner J.H. Abrahamson D.R. Sanes J.R. Kasinath B.S. J. Am. Soc. Nephrol. 1999; 10: 1931-1939Crossref PubMed Google Scholar). Mice were sacrificed at the end of each experimental period, and renal cortex was dissected out and processed for further analysis. Statistical Analysis—All values are expressed as mean ± S.E. obtained from at least three independent experiments. Statistical analysis was performed using one-way analysis of variance (ANOVA) for comparison between multiple groups and posthoc analysis using Student's Newman Keul's multiple comparison tests using Graph Pad Prism 4 software. Statistical comparisons between two groups were performed by the Student's t test. Statistical significance was assigned to values of p < 0.05. High Glucose and High Insulin Stimulate Laminin and Fibronectin Synthesis—Incubation of MCT cells with 30 mm glucose or 1 nm insulin stimulated synthesis of extracellular matrix proteins, laminin β1, and fibronectin, starting at 6 h and reaching a peak at 24 h (Fig. 1, A–D). We investigated if induction of synthesis of laminin β1 and fibronectin was due to an increase in their mRNA levels. Quantitative real time RT-PCR showed an increase in their respective mRNA content upon treatment with high glucose and high insulin (Fig. 2, A–D). Preincubation with actinomycin D, a transcription inhibitor, inhibited laminin β1 synthesis induced by high glucose and high insulin at 24 h but not at 1 and 2 h, suggesting transcriptional regulation at later time points and, possibly, translational regulation at early time points. Actinomycin-D inhibited high glucose- and high insulin-induced fibronectin synthesis at 2 and 24 h but not at 1 h, suggesting transcriptional regulation beyond 1 h of incubation (supplemental Fig. S1). We have previously reported that high glucose, high insulin, high glucose + high insulin increased de novo protein synthesis by about 20–25% (27Mariappan M.M. Feliers D. Mummidi S. Choudhury G.G. Kasinath B.S. Diabetes. 2007; 56: 476-485Crossref PubMed Scopus (67) Google Scholar). Immunoprecipitation of [35S]methionine-labeled cell protein with lamininβ1 antibody showed significant increment in laminin synthesis under the three conditions. These data showed that although high glucose, high insulin, and both together stimulated global protein synthesis in MCT cells, increments in laminin β1 synthesis were in addition individually stimulated (27Mariappan M.M. Feliers D. Mummidi S. Choudhury G.G. Kasinath B.S. Diabetes. 2007; 56: 476-485Crossref PubMed Scopus (67) Google Scholar).FIGURE 2High glucose and high insulin increase laminin β1 and fibronectin mRNA levels. Total RNA isolated from cells treated with or without high glucose and high insulin was reverse-transcribed to synthesize cDNA. 2μl of the cDNA subjected to quantitative real-time PCR using the SYBR Green method shows an increase in the transcript level of lamininβ1(A and B) and fibronectin (C and D) following incubation with high glucose or high insulin. GAPDH served as an internal control. The ratio of laminin β1 and fibronectin mRNA to GAPDH mRNA is shown. Histograms represent means ± S.E. from three independent experiments. Significant differences between control and cells treated with high glucose and high insulin are indicated by #, p < 0.05; §, p < 0.01; and *, p < 0.001 by ANOVA.View Large Image Figure ViewerDownload Hi-res image Download (PPT) High Glucose and High Insulin Promote Phosphorylation and Inactivation of GSK3β and Dephosphorylation of eIF2Bϵ—In cells in the basal state, GSK3β is active and keeps eIF2Bϵ inactive. Both high glucose and high insulin induced phosphorylation of Ser-9 on GSK3β, which is required for its inactivation, starting at about 2 h and lasting up to 48 h (Fig. 3, A and B). Combined incubation with high glucose and high insulin also increased GSK3β phosphorylation following the same time course as that of high glucose alone or high insulin alone (supplemental Fig. S2). We directly studied the effect of high glucose and high insulin on GSK3β activity. In an immunokinase activity assay, treatment of MCT cells with high glucose and high insulin significantly reduced [32P]phosphate incorporation into the GSK3β substrate, demonstrating reduced activity of GSK3β (Fig. 3, C and D). These data show that the increase in Ser-9 phosphorylation of GSK3β correlates with reduction in its enzymatic activity. eIF2Bϵ mediates the antihypertrophic effects of GSK3β. Upon inactivation of GSK3β eIF2Bϵ is dephosphorylated on Ser-539 and is free to promote translation initiation. The binding of eIF2 to the activated initiator methionyl tRNA is promoted by dephosphorylated eIF2Bϵ (34Kasinath B.S. Mariappan M.M. Sataranatarajan K. Lee M.J. Feliers D. J. Am. Soc. Nephrol. 2006; 17: 3281-3292Crossref PubMed Scopus (52) Google Scholar, 35Proud C.G. Biochem. J. 2007; 403: 217-234Crossref PubMed Scopus (402) Google Scholar). High glucose and high insulin reduced eIF2Bϵ Ser-539 phosphorylation starting at 2 h and lasting for up to 48 h (Fig. 3, E and F). Thus, GSK3β inactivation and eIF2Bϵ dephosphorylation are temporally associated with high glucose- and high insulin-induced matrix protein synthesis. High Glucose and High Insulin Regulate Initiation and Elongation Phases of Translation—Because translation is the rate-limiting step in gene expression culminating in peptide synthesis, we examined key steps in initiation and elongation phases of translation in the context of GSK3β regulation of matrix protein synthesis. During the initiation phase of mRNA translation, binding of the 43S ribosomal pre-initiation complex to the mRNA is facilitated by eIF4E in association with eIF4G and eIF4A (15Kasinath B.S. Lee M.J. Feliers D. Sonenberg N. Cortes P.M.C. Contemporary Diabetes. The Diabetic Kidney. Humana Press, NJ2003: 97-116Google Scholar). In the resting cell, eIF4E is held in an inactive complex by its binding protein 4E-BP1. Upon stimulation, 4E-BP1 is phosphorylated on several threonine and serine residues, including Thr-37/46 (36Gingras A.C. Gygi S.P. Raught B. Polakiewicz R.D. Abraham R.T. Hoekstra M.F. Aebersold R. Sonenberg N. Genes Dev. 1999; 13: 1422-1437Crossref PubMed Scopus (1004) Google Scholar). This results in dissolution of the complex, freeing eIF4E to bind with mRNA cap to promote translation initiation. High glucose and high insulin augmented phosphorylation of 4E-BP1 (Fig. 4, A and B) and eIF4E (Fig. 4, C and D). During the elongation phase of translation, amino acids are sequentially added to the nascent peptide chain by the participation of aminoacyl tRNA (34Kasinath B.S. Mariappan M.M. Sataranatarajan K. Lee M.J. Feliers D. J. Am. Soc. Nephrol. 2006; 17: 3281-3292Crossref PubMed Scopus (52) Google Scholar, 37Thornton S. Anand N. Purcell D. Lee J. J. Mol. Med. 2003; 81: 536-548Crossref PubMed Scopus (116) Google Scholar). Movement of the ribosome along the mRNA, resulting in translocation of aminoacyl tRNA from the A site to the P site on the ribosome, is regulated by eukaryotic elongation factor 2 (eEF2). Activation of eEF2 occurs following dephosphorylation at Thr-56 (38Redpath N.T. Price N.T. Severinov K.V. Proud C.G. Eur. J. Biochem. 1993; 213: 689-699Crossref PubMed Scopus (155) Google Scholar). High glucose and high insulin also induced dephosphorylation of Thr-56 on eEF2 (Fig. 4, E and F). Thus, high glucose or high insulin activate important steps in both initiation and elongation phases in association with inactivation of GSK3β and increased matrix protein synthesis in MCT cells. High Glucose and High Insulin Activate Akt, Erk and p70S6 Kinase—Several upstream kinases have been implicated in inactivation of GSK3β by phosphorylation at Ser-9 (39Cohen P. Frame S. Nat. Rev. Mol. Cell. Biol. 2001; 2: 769-776Crossref PubMed Scopus (1298) Google Scholar). These include Akt-PKB (40Haq S. Choukroun G. Kang Z.B. Ranu H. Matsui T. Rosenzweig A. Molkentin J.D. Alessandrini A. Woodgett J. Hajjar R. Michael A. Force T. J. Cell Biol. 2000; 151: 117-130Crossref PubMed Scopus (335) Google Scholar), Erk-1/-2 MAP kinase, and p70S6 kinase (41Cross D.A. Alessi D.R. Vandenheede J.R. McDowell H.E. Hundal H.S. Cohen P. Biochem. J. 1994; 303: 21-26Crossref PubMed Scopus (420) Google Scholar, 42Zhang H.H. Lipovsky A.I. Dibble C.C. Sahin M. Manning B.D. Mol. Cell. 2006; 24: 185-197Abstract Full Text Full Text PDF PubMed Scopus (247) Google Scholar). We immunoblotted cell lysates treated with phosphospecific antibodies for Akt, Erk, and p70S6 kinase phosphorylated at Ser-473, Thr-202/Tyr-204, and, Thr-389, respectively. High glucose and high insulin stimulated phosphorylation of Akt, Erk, and p70S6 kinase that started at 2 h and remained phosphorylated until 24–48 h. (Fig. 5, A–F). Akt but Not p70S6 Kinase Regulates GSK3β Phosphorylation Induced by High Glucose and High Insulin—Next, we examined whether phosphorylation of Akt and p70S6 kinase is required for high glucose- and high insulin-induced phosphorylation of GSK3β and laminin β1 synthesis. In cells expressing dominant-negative Akt, high glucose and high insulin failed to stimulate GSK3β phosphorylation and laminin β1 synthesis (Fig. 6, A and B). However, in cells expressing kinasedead p70S6 kinase, only laminin β1 synthesis was blocked but not Ser-9 phosphorylation of GSK3β (Fig. 6, C and D). Thus, stimulation of GSK3β phosphorylation by high glucose and high insulin is dependent on Akt but not p70S6 kinase activation in MCT cells, although both kinases are involved in the regulation of laminin β1 synthesis. We examined the role of PI 3-kinase, Erk, and mTOR in GSK3β Ser-9 phosphorylation. Akt activation by high glucose and high insulin is PI 3-kinase-dependent (data not shown) (43King W.G. Mattaliano M.D. Chan T.O. Tsichlis P.N. Brugge J.S. Mol. Cell. Biol. 1997; 17: 4406-4418Crossref PubMed Scopus (385) Google Scholar) and Akt activation results in GSK3β inhibition by phosphorylation of Ser-9 (44Jope R.S. Johnson G.V. Trends Biochem. Sci. 2004; 29: 95-102Abstract Full Text Full Text PDF PubMed Scopus (1325) Google Scholar). Preincubation of MCT cells with LY294002, a selective inhibitor of PI 3-kinase, U0126, a MEK inhibitor, and, rapamycin, a specific inhibitor of mTOR, blocked GSK3β phosphorylation (Fig. 7, A–F) and laminin β1 synthesis (Fig. 7, G and H) induced by high glucose and high insulin at 24 h. As rapamycin data suggested, mTOR regulates GSK3β, we studied their interaction. Immunoprecipitation with anti-mTOR antibody and immunoblotting with anti-GSK3β antibody suggested that the two proteins exist in a complex; the intensity of binding did not seem to change following exposure to high glucose or high insulin (supplemental Fig. S3). Because PI 3-kinase-Akt-mTOR belong to a linear pathway, it is not surprising that inhibitors of PI 3-kinase (LY294002) and mTOR (rapamycin) inhibited GSK3β phosphorylation. We explored if Erk activation was under the regulation of PI 3-kinase. LY294002, abrogated high glucose- and high insulin-induced phosphorylation of Erk1/2 MAP kinase (supplemental Fig. S4), suggesting the latter was under the control of PI 3-kinase. These data confirm our previous report that Erk is downstream of PI 3-kinase in insulin-treated MCT cells (45Bhandari B.K. Feliers D. Duraisamy S. Stewart J.L. Gingras A.C. Abboud H.E. Choudhury G.G. Sonenberg N. Kasinath B.S. Kidney Int. 2001; 59: 866-875Abstract Full Text Full Text PDF PubMed Scopus (70) Google Scholar). Because PI 3-kinase regulates both mTOR and Erk activation, we see complete inhibition of Ser-9 phosphorylation of GSK3β with each of the three inhibitors. Expression of Kinase Inactive (K85A) and Constitutively Active (S9A) Constructs of GSK3β Alter High Glucose- and High Insulin-induced Changes in Laminin β1 Synthesis—Next, we investigated the requirement of GSK3β inactivation for high glucose- and high insulin-induced synthesis of laminin β1 in MCT cells. Cells were transiently transfected with either a kinase inactive (K85A) or a constitutively active (S9A) construct of GSK3β. Expression of kinase-dead K85A-GSK3β resulted in increased laminin β1 synthesis in control cells (Fig. 8, A and B). However, inactive GSK3β did not add to the incremental effect of high glucose and high insulin on laminin β1 syn" @default.
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• W2004608929 title "Glycogen Synthase Kinase 3β Is a Novel Regulator of High Glucose- and High Insulin-induced Extracellular Matrix Protein Synthesis in Renal Proximal Tubular Epithelial Cells" @default.
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