TDAG51 Mediates the Effects of Insulin-like Growth Factor I (IGF-I) on Cell Survival
2004; Elsevier BV; Volume: 279; Issue: 24 Linguagem: Inglês
10.1074/jbc.m400661200
ISSN1083-351X
AutoresYuka Toyoshima, Michael Karas, Shoshana Yakar, Joëlle Dupont, Lee J. Helman, Derek LeRoith,
Tópico(s)Growth Hormone and Insulin-like Growth Factors
ResumoInsulin-like growth factor-I (IGF-I) receptors and insulin receptors belong to the same subfamily of receptor tyrosine kinases and share a similar set of intracellular signaling pathways, despite their distinct biological actions. In the present study, we evaluated T cell death-associated gene 51 (TDAG51), which we previously identified by cDNA microarray analysis as a gene specifically induced by IGF-I. We characterized the signaling pathways by which IGF-I induces TDAG51 gene expression and the functional role of TDAG51 in IGF-I signaling in NIH-3T3 (NWTb3) cells, which overexpress the human IGF-I receptor. Treatment with IGF-I increased TDAG51 mRNA and protein levels in NWTb3 cells. This effect of IGF-I was specifically mediated by the IGF-IR, because IGF-I did not induce TDAG51 expression in NIH-3T3 cells overexpressing a dominant-negative IGF-I receptor. Through the use of specific inhibitors of various protein kinases, we found that IGF-I induced TDAG51 expression via the p38 MAPK pathway. The ERK, JNK, and phosphatidylinositol 3-kinase pathways were not involved in IGF-I-induced regulation of TDAG51. To assess the role of TDAG51 in IGF-I signaling, we used small interfering RNA (siRNA) expression vectors directed at two different target sites to reduce the level of TDAG51 protein. In cells expressing these siRNA vectors, TDAG51 protein levels were decreased by 75-80%. Furthermore, TDAG51 siRNA expression abolished the ability of IGF-I to rescue cells from serum starvation-induced apoptosis. These findings suggest that TDAG51 plays an important role in the anti-apoptotic effects of IGF-I. Insulin-like growth factor-I (IGF-I) receptors and insulin receptors belong to the same subfamily of receptor tyrosine kinases and share a similar set of intracellular signaling pathways, despite their distinct biological actions. In the present study, we evaluated T cell death-associated gene 51 (TDAG51), which we previously identified by cDNA microarray analysis as a gene specifically induced by IGF-I. We characterized the signaling pathways by which IGF-I induces TDAG51 gene expression and the functional role of TDAG51 in IGF-I signaling in NIH-3T3 (NWTb3) cells, which overexpress the human IGF-I receptor. Treatment with IGF-I increased TDAG51 mRNA and protein levels in NWTb3 cells. This effect of IGF-I was specifically mediated by the IGF-IR, because IGF-I did not induce TDAG51 expression in NIH-3T3 cells overexpressing a dominant-negative IGF-I receptor. Through the use of specific inhibitors of various protein kinases, we found that IGF-I induced TDAG51 expression via the p38 MAPK pathway. The ERK, JNK, and phosphatidylinositol 3-kinase pathways were not involved in IGF-I-induced regulation of TDAG51. To assess the role of TDAG51 in IGF-I signaling, we used small interfering RNA (siRNA) expression vectors directed at two different target sites to reduce the level of TDAG51 protein. In cells expressing these siRNA vectors, TDAG51 protein levels were decreased by 75-80%. Furthermore, TDAG51 siRNA expression abolished the ability of IGF-I to rescue cells from serum starvation-induced apoptosis. These findings suggest that TDAG51 plays an important role in the anti-apoptotic effects of IGF-I. IntroductionInsulin-like growth factor-I (IGF-I) 1The abbreviations used are: IGF-I, insulin-like growth factor-I; IGF-IR, IGF-I receptor; IR, insulin receptor; PI3K, phosphatidylinositol 3-kinase; JNK, c-Jun NH2-terminal kinase; ERK, extracellular signal-regulated kinase; MAPK, mitogen-activated protein kinase; MEK, mitogen-activated protein kinase/extracellular signal-regulated kinase kinase; PARP, poly(ADP-ribose) polymerase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; siRNA, small interfering RNA; FBS, fetal bovine serum; 7-AAD, 7-aminoactinomycin D. is a polypeptide hormone that is structurally homologous to insulin. The pleiotrophic effects of IGF-I and insulin on cell proliferation, cell survival, and metabolism are mediated by a complex network of intracellular signaling pathways. The biological and physiological functions of IGF-I and insulin are initiated when these ligands bind to their receptors. The structures of the IGF-I receptor (IGF-IR) and insulin receptor (IR) are similar, each consisting of two extracellular α-subunits and two transmembrane β-subunits. Although both ligands interact with each receptor, the receptors bind their own ligands with 100-1000-fold higher affinity than that of the heterologous peptides. After ligand binding, each receptor becomes autophosphorylated and the intrinsic tyrosine kinase activity of these receptors becomes activated. Various substrate proteins, including Shc, Gab-1, and the insulin receptor substrate proteins, are then phosphorylated on tyrosine residues by the activated receptors. Tyrosine-phosphorylated insulin receptor substrate and Shc molecules interact with specific downstream signaling molecules containing Src homology 2 domains, including the p85 regulatory subunit of phosphatidylinositol 3-kinase (PI3K) and Grb2, which lead to activation of the PI3K pathway and Ras/Raf/MAPK signaling pathways, respectively (1LeRoith D. Werner H. Beitner-Johnson D. Roberts Jr., C.T. Endocr. Rev. 1995; 16: 143-163Crossref PubMed Scopus (1241) Google Scholar, 2Adamo M. Roberts Jr., C.T. LeRoith D. Biofactors. 1992; 3: 151-157PubMed Google Scholar, 3Dupont J. LeRoith D. Horm. Res. 2001; 55: 22-26Crossref PubMed Scopus (169) Google Scholar). Although the IGF-IR and IR signaling pathways generally overlap, IGF-I and insulin exhibit distinct physiological functions. Whereas insulin generally regulates metabolism, IGF-I controls cell growth, differentiation, and protects cells against apoptosis (4Baserga R. Hongo A. Rubini M. Prisco M. Valentinis B. Biochim. Biophys. Acta. 1997; 1332: F105-F126Crossref PubMed Scopus (482) Google Scholar, 5Vincent A.M. Feldman E.L. Growth Horm. IGF Res. 2002; 12: 193-197Crossref PubMed Scopus (235) Google Scholar). The differences between IGF-I and insulin receptor signaling that mediate these distinct biological effects remain to be elucidated.In a previous study, we stimulated NIH-3T3 fibroblasts either with IGF-I or with insulin and then evaluated changes in gene expression patterns by cDNA microarray analysis to identify genes that are differentially regulated by IGF-I and insulin (6Dupont J. Khan J. Qu B.H. Metzler P. Helman L. LeRoith D. Endocrinology. 2001; 142: 4969-4975Crossref PubMed Scopus (88) Google Scholar). We found 30 genes that were specifically responsive to IGF-I. Most of these were related to mitogenesis and differentiation. We characterized Twist, which is one of the genes that are specifically responsive to IGF-I, and showed that Twist is positively involved in the anti-apoptotic effects of IGF-I (7Dupont J. Fernandez A.M. Glackin C.A. Helman L. LeRoith D. J. Biol. Chem. 2001; 276: 26699-26707Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar). Here, we have evaluated TDAG51 (T cell death-associated gene 51), another gene that is specifically regulated by IGF-I.Mouse TDAG51 was originally isolated and shown to regulate the expression of Fas and T cell receptor activation-induced apoptosis in mouse T cell hybridomas (8Park C.G. Lee S.Y. Kandala G. Choi Y. Immunity. 1996; 4: 583-591Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). TDAG51 is ubiquitously expressed in mice, and strong expression is found in brain, lung, liver, and thymus (8Park C.G. Lee S.Y. Kandala G. Choi Y. Immunity. 1996; 4: 583-591Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar). Subsequently, the rat and human homologues were readily identified. The rat homologue was isolated as an immediate early gene induced by fibroblast growth factor in neuronal cells and was shown to promote cell death (9Gomes I. Xiong W. Miki T. Rosner M.R. J. Neurochem. 1999; 73: 612-622Crossref PubMed Scopus (57) Google Scholar). The human homologue was shown to be down-regulated in metastatic melanoma cells, as compared with primary melanoma cells (10Neef R. Kuske M.A. Prols E. Johnson J.P. Cancer Res. 2002; 62: 5920-5929PubMed Google Scholar). The TDAG51 protein has highly repeated sequences in its carboxyl-terminal region, including prolineglutamine (PQ) repeats and proline-histidine (PH) repeats. It has been shown that proteins containing PQ-rich domains may function as transcriptional activators and mediate apoptosis in various neurodegenerative diseases, such as Huntington's disease (11Li J.Y. Plomann M. Brundin P. Trends Mol. Med. 2003; 9: 414-420Abstract Full Text Full Text PDF PubMed Scopus (154) Google Scholar). Taken together, several lines of evidence suggest that TDAG51 may be associated with enhanced apoptosis.It has been well established that IGF-I can protect cells from apoptosis under a variety of circumstances. For example, IGF-I prevents apoptosis induced by overexpression of c-myc in fibroblasts (12Harrington E.A. Bennett M.R. Fanidi A. Evan G.I. EMBO J. 1994; 13: 3286-3295Crossref PubMed Scopus (732) Google Scholar), by interleukin-3 withdrawal in interleukin-3-dependent hemopoietic cells (13McCubrey J.A. Steelman L.S. Mayo M.W. Algate P.A. Dellow R.A. Kaleko M. Blood. 1991; 78: 921-929Crossref PubMed Google Scholar, 14Rodriguez-Tarduchy G. Collins M.K. Garcia I. Lopez-Rivas A. J. Immunol. 1992; 149: 535-540PubMed Google Scholar), by the topoisomerase I inhibitor, etoposide (15Sell C. Baserga R. Rubin R. Cancer Res. 1995; 55: 303-306PubMed Google Scholar), by anti-cancer drugs (16Dunn S.E. Hardman R.A. Kari F.W. Barrett J.C. Cancer Res. 1997; 57: 2687-2693PubMed Google Scholar), by irradiation with UV-B (17Kulik G. Klippel A. Weber M.J. Mol. Cell. Biol. 1997; 17: 1595-1606Crossref PubMed Scopus (964) Google Scholar), and by serum deprivation in PC12 cells (18Parrizas M. Saltiel A.R. LeRoith D. J. Biol. Chem. 1997; 272: 154-161Abstract Full Text Full Text PDF PubMed Scopus (605) Google Scholar). While the anti-apoptotic effect of IGF-I has been clearly demonstrated, the molecular mechanisms by which IGF-I inhibits apoptosis induced by these various stimuli remain unknown.In this study, we set out to determine the signaling pathways involved in IGF-I-induced expression of TDAG51 in NIH-3T3 cells and to determine the role of TDAG51 in the functions of IGF-I in these cells.EXPERIMENTAL PROCEDURESMaterials—Human recombinant IGF-I was a gift from Genentech (South San Francisco, CA). LY294002, which is a PI3K-specific inhibitor, was purchased from Sigma. U0126, a MEK1/2-specific inhibitor and SB202190, a p38 MAPK-specific inhibitor were obtained from Calbiochem. SP600215, a JNK-specific inhibitor, was from Biomol Research Laboratories Inc. (Plymouth Meeting, MA). The stock solutions of these inhibitors were prepared in Me2SO at a 1000-fold concentration, such that the concentration of Me2SO was below 0.1% when the compounds were added to the culture medium. Polyclonal antibodies to TDAG51 (M-20) and to the IGF-IR β-subunit (C-20) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA). A monoclonal antibody to actin was obtained from Sigma. Anti-phospho-p38 MAPK, anti-p38 MAPK, anti-phospho-Akt (Ser-473), anti-Akt, anti-ERK1/2, anti-phospho-ERK, and anti-cleaved poly(ADP-ribose) polymerase (PARP) antibodies were purchased from Cell Signaling Technology (Beverly, MA). A monoclonal antibody to the human IGF-IR (αIR-3) was obtained from Oncogene Research Products (Cambridge, MA). Mouse IgG was from Pierce. The pTRI-GAPDH-mouse antisense control template was purchased from Ambion (Austin, TX). The radionuclide [α-32P]dCTP (6000 Ci/mmol) was from PerkinElmer Life Sciences.Construction of Expression Plasmids—The pSilencer™ 1.0-U6 siRNA expression vector was purchased from Ambion (Austin, TX). As the inserts for expressing short hairpin RNA, two inserts were selected: 21-sense (5′-GCAGCTACAACAGCAGCAGTTCAAGAGACTGCTGCTGTTGTAGCTGCTTTTTT-3′) and 21-antisense (5′-AATTAAAAAAGCAGCTACAACAGCAGCAGTCTCTTGAACTGCTGCTGTTGTAGCTGCGGCC-3′) and 37-sense (5′-GTCTACCAGGCAGAAGCAGTTCAAGAGACTGCTTCTGCCTGGTAGACTTTTTT-3′) and 37-antisense (5′-AATTAAAAAAGTCTACCAGGCAGAAGCAGTCTCTTGAACTGCTTCTGCCTGGTAGACGGCC-3′). Each insert was annealed and subcloned with pSilencer, which was linearized with ApaI and EcoRI. The construct containing siRNA insert 21 or 37 was designated psi21 or psi37. We determined whether the transfected clones contained the siRNA expression vectors by PCR analysis. The forward and reverse primers were 5′-GATCTTGTGGGAGAAGCTCGGCT-3′ and 5′-ACAAAAGCTGGAGCTCCACCGC-3′, respectively. Genomic DNA was isolated from each cell line using DNeasy Tissue Kit (Qiagen, Valencia, CA).Cell Culture and Stable Transfection—The NWTb3 cell line expresses the human IGF-IR at a level of ∼4 × 105 receptors per cell (19Blakesley V.A. Kato H. Roberts Jr., C.T. LeRoith D. J. Biol. Chem. 1995; 270: 2764-2769Abstract Full Text Full Text PDF PubMed Scopus (32) Google Scholar). Two cell lines expressing dominant-negative forms of the human IGF-IR (∼3-7 × 105 receptors/cell) were also used. These include the NKA8 cell line, in which the Lys-1003 residue at the ATP-binding site was substituted with Ala (NKA8 mutant), and the NKR1 cell line, in which Lys-1003 was substituted with Arg (NKR1 mutant) (20Kato H. Faria T.N. Stannard B. Roberts Jr., C.T. LeRoith D. J. Biol. Chem. 1993; 268: 2655-2661Abstract Full Text PDF PubMed Google Scholar). The IR cell line, a gift from Dr. S. Taylor (National Institues of Health, Bethesda, MD), expresses the human IR at a level of about 2 × 106 receptor/cell (21Levy-Toledano R. Accili D. Taylor S.I. Biochim. Biophys. Acta. 1993; 1220: 1-14Crossref PubMed Scopus (14) Google Scholar). NWTb3, NKR1, NKA8, and IR cells were cultured in Dulbecco's modified Eagle's medium supplemented with 10% heat-inactivated fetal bovine serum (FBS), 100 units/ml penicillin, 100 μg/ml streptomycin, 300 mg/ml l-glutamine, and Geneticin (0.5 g/liter, Invitrogen) in a humidified atmosphere of 95% air and 5% CO2 at 37 °C.For stable transfections, NWTb3 cells were grown to 70-80% confluence in complete culture medium. The cells were harvested by trypsinization and suspended in complete culture medium at 1 × 107 cells/ml. In a 0.4-cm cuvette, 0.4 ml of the cell suspension was mixed with the TDGA51 siRNA expression vector (psi21 and psi37) or pSilencer (30 μg) and pcDNA3.1-hygro (+) (10 μg, Invitrogen). The samples were then electroporated in a Bio-Rad Gene Pulsar (Bio-Rad) at 950 microfarads and 0.22 kV/cm (t = 20-30 ms). After incubation at room temperature for 10 min, the electroporated cells were diluted (1:100) in complete culture medium and plated into 100-mm dishes (22Worley B.S. van den Broeke L.T. Goletz T.J. Pendleton C.D. Daschbach E.M. Thomas E.K. Marincola F.M. Helman L.J. Berzofsky J.A. Cancer Res. 2001; 61: 6868-6875PubMed Google Scholar). Beginning 48 h after transfection, 0.2 g/liter of hygromycin (Clontech) was added to the cultures to select for clones expressing siRNA. Two weeks later, independent colonies were picked using coloning disks (Scienceware). The resulting stable clones (siHygro1, si21-3, and si37-57) were cultured in complete culture medium with Hygromycin (0.2 g/liter).Northern Blot Analysis—The template for the TDAG51 cDNA probe was obtained by RT-PCR. The position of the amplified cDNA was as follows: mouse TDAG51 (GenBank™ accession number NM_009344) 552-851. The amplified cDNA was subcloned into the pCR®II-TOPO vector (Invitrogen), and the resulting plasmid was subjected to DNA sequencing analysis to confirm the sequence. The TDAG51 cDNA template was labeled using the Rediprime labeling kit (Amersham Biosciences).Total RNA was isolated using TRIzol reagent (Invitrogen) according to the manufacturer's recommended instructions. Total RNA (20 μg) was resolved on 1.25% denaturing agarose gels. The integrity and amount of RNA were confirmed by visualization of ribosomal RNA. After electrophoresis, RNA was transferred to Nytran nylon membranes (Schleicher & Schüll) by capillary action overnight and immobilized by UV exposure. Blots were prehybridized for 2 h at 42 °C in a buffer containing 50% formamide, 5× Denhardt's, 1% SDS, 5× SSC, and 100 μl/ml salmon sperm. Blots were then hybridized overnight at the same temperature with 5× 106 cpm/ml [32P]dCTP-labeled DNA probe in a buffer containing 50% formamide, 2.5× Denhardt's, 1% SDS, 5× SSC, and 100 μl/ml salmon sperm. The blots were washed at high stringency and the hybridized radioactivity was measured using Fuji BAS1800II instrument (FujiFilm, Stamford, CT). TDAG51 mRNA levels were quantified and normalized to GAPDH levels, using the Image Reader software and Image Gauge software together with a Fuji BAS1800II instrument.Western Blot Analysis—Cell lysates were prepared in lysis buffer (10 mm Tris, pH 7.4, 150 mm NaCl, 1 mm EDTA, 1% Triton-X, 100 mm sodium fluoride, 10 mm sodium orthovanadate, 10 mm sodium pyrophosphate) to which a protease inhibitor mixture was added (Complete Mini EDTA-free, Roche Applied Science). Lysates were centrifuged at 12,000 × g for 30 min at 4 °C to remove insoluble materials. The protein concentration in the supernatants was determined with the BCA protein assay kit (Pierce). The extracted protein samples were subjected to SDS-PAGE and transferred to nitrocellulose membranes (Invitrogen). The membranes were blocked with 5% nonfat milk in Tris-buffered saline with 0.1% Tween 20 (TBS-T) for 1 h at room temperature. The membranes were then incubated with various antibodies overnight, as indicated in the figure legends. After washing with TBS-T, the membranes were incubated with the appropriate horseradish peroxidase-conjugated secondary antibodies (Amersham Biosciences) for 1 h and washed again. Immunoreactivity was detected with an enhanced chemiluminescence kit (PerkinElmer Life Sciences) and quantified by densitometry, using Mac Bas V2.52 software (FujiFilm, Stamford, CT).Analysis of Apoptosis by Flow Cytometry—Cells were plated on 100-mm dishes in the culture medium. After 18 h of incubation, the medium was changed to serum-free medium with or without IGF-I (50 nm), and the cells were incubated for another 48 h. For the treatment with mouse IgG or αIR3 antibody, the cells were preincubated in serum-free medium with mouse IgG or αIR-3 antibody (1.0 μg/ml) for 2 h and then FBS (10%) and IGF-I (10 nm) was added or not to the medium for another 48 h. The cells were then collected and washed twice with HEPES Buffer (10 mm HEPES/NaOH, pH 7.4, 140 mm NaCl, and 2.5 mm CaCl2) at room temperature as described previously (23Karas M. Zaks T.Z. Liu J.L. LeRoith D. Mol. Biol. Cell. 1999; 10: 4441-4450Crossref PubMed Scopus (20) Google Scholar). Cells were resuspended in 0.2 ml of HEPES Buffer that included 3 μl of Annexin V-FITC (Pharmingen) and 5 μl of 7-aminoactinomycin D (7-AAD) (Pharmingen) and were incubated for 15 min at room temperature in the dark. Finally, the stained cells were analyzed by FACSCalibur using CellQuest Software (BD Biosciences).Analysis of the Cleaved Caspase-3 and PARP—Cells were plated on 100-mm dishes in the culture medium. After 18 h, the medium was changed to serum-free Dulbecco's modified Eagle's medium with or without IGF-I (50 nm), and the cells were incubated for another 48 h. The cells were then collected and subjected to lysis as described above. The cleavage of caspase-3 and PARP was analyzed by subjecting cell lysates to immunoblotting with anti-cleaved caspase-3 and PARP antibodies.Statistical Analyses—All values are expressed as the mean ± S.E. The statistical significance was determined by unpaired Student's t test using Statview 5.0 software (SAS Institute Inc., Cary, NC). Differences were considered to be statistically significant at p < 0.05.RESULTSTDAG51 Expression Is Induced by IGF-I in NWTb3 Cells—In a previous cDNA microarray analysis study, we showed that TDAG51 gene expression was specifically induced by IGF-I (6Dupont J. Khan J. Qu B.H. Metzler P. Helman L. LeRoith D. Endocrinology. 2001; 142: 4969-4975Crossref PubMed Scopus (88) Google Scholar). To confirm this finding, we tested the effects of various durations of IGF-I treatment (50 nm) on TDAG51 mRNA levels in NWTb3 cells. The effect of IGF-I on TDAG51 mRNA was maximal after 1.5 h of stimulation, which increased TDAG51 levels by 10-fold. After longer incubation times (3-24 h), TDAG51 gene expression progressively decreased (Fig. 1A). We also analyzed TDAG51 protein levels and showed TDAG51 protein was increased ∼6-fold by IGF-I after 3-6 h of stimulation. This high level of protein expression persisted through 12 h of IGF-I stimulation (Fig. 1B). TDAG51 protein level gradually decreased after 12 h of IGF-I stimulation, and the IGF-I effect was abolished after 36 h of IGF-I treatment. Thus, IGF-I strongly stimulates the expression of both TDAG51 mRNA and protein in NWTb3 cells. On the other hand, in NIH-3T3 cells overexpressing human insulin receptors (IR cells), insulin's effect (50 nm) on TDAG51 gene expression was only 2-fold after 1.5 h of stimulation (data not shown).To confirm that IGF-I induced TDAG51 expression was mediated through the IGF-IR, we used NKR1 and NKA8 cells, which overexpress dominant-negative versions of the human IGF-IR in NIH-3T3 cells. NKR1 and NKA8 cells were stimulated with IGF-I (50 nm) for 1.5 h, and TDAG51 gene expression was determined by Northern blot analysis. Fig. 2 shows that the increase in TDAG51 induced by IGF-I was only observed in NWTb3 cells (which overexpress the wild-type human IGF-IR) and not in either the NKR1 or NKA cell lines. These results suggest that activation of the IGF-IR is essential for IGF-I-induced TDAG51 expression.Fig. 2The effect of IGF-IR activation on TDAG51 expression in mouse fibroblasts. NWTb3, NKR1, and NKA8 cells (70-80% confluent) were serum-starved overnight and then incubated with or without IGF-I (50 nm) for 1.5 h, as indicated. Total RNA was isolated and analyzed by Northern blot analysis to measure TDAG51 mRNA levels (upper panel). After autoradiography and quantification, membranes were stripped and reprobed with a mouse GAPDH probe, as an internal control. Data are expressed as the mean ± S.E. for three separate experiments (lower panel).View Large Image Figure ViewerDownload Hi-res image Download (PPT)The p38 MAPK Pathway Is Involved in IGF-I-induced TDAG51 Expression in NWTb3 Cells—To delineate the signaling pathways involved in IGF-I-induced expression of TDAG51, we used specific inhibitors for various protein kinases known to be activated by IGF-I, including PI3K, p38 MAPK, ERK1/2, and JNK1/2. We first evaluated the activity of these inhibitors in our system. NWTb3 cells were treated with the following inhibitors: 50 μm LY294002, a specific inhibitor of PI3K, 50 μm SB202190, a specific inhibitor of p38 MAPK, 2 μm U0126, a specific inhibitor of MEK, or 20 μm SP600125, a specific inhibitor of JNK1/2, for 1 h prior to stimulation with IGF-I (50 nm, 10 min). Western blot analysis was performed using antibodies against the phospho- and total forms of Akt, p38 MAPK, ERK1/2, and JNK, as described previously (7Dupont J. Fernandez A.M. Glackin C.A. Helman L. LeRoith D. J. Biol. Chem. 2001; 276: 26699-26707Abstract Full Text Full Text PDF PubMed Scopus (91) Google Scholar, 24Heron-Milhavet L. Karas M. Goldsmith C.M. Baum B.J. LeRoith D. J. Biol. Chem. 2001; 276: 18185-18192Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Each inhibitor effectively prevented the IGF-I-induced phosphorylation of each of these protein kinases (data not shown). We next analyzed the effects of these inhibitors on IGF-I-induced expression of TDAG51. NWTb3 cells were pretreated with these protein kinase inhibitors for 1 h and prior to stimulation with IGF-I (50 nm for 1.5 h). Total RNA was then isolated and subjected to Northern blot analysis, as described under "Experimental Procedures." As shown in Fig. 3, pretreatment with LY294002, U0126, and SP600125 did not significantly alter the effects of IGF-I on TDAG51 expression. However, pretreatment with SB202190 blocked IGF-I-induced TDAG51 expression by 80%. These results suggest that the effects of IGF-I on TDAG51 gene expression are mediated primarily by the p38 MAPK pathway.Fig. 3The effect of several protein kinase inhibitors on IGF-I-induced TDAG51 expression in NWTb3 cells. Serum-starved NWTb3 cells were preincubated for 1 h with the following inhibitors: 50 μm LY294002, 50 μm SB202190, 2 μm U0126, or 20 μm SP600125. Cells were then stimulated with or without IGF-I (50 nm) for 1.5 h, as indicated. Total RNA was isolated and analyzed by Northern blot analysis to measure TDAG51 mRNA levels (upper panel). After autoradiography and quantification, membranes were stripped and reprobed with a mouse GAPDH probe, as an internal control. Data are expressed as the mean ± S.E. for three separate experiments (lower panel). *, p < 0.05 versus non-IGF-I-simulated cells in NWTb3 cells.View Large Image Figure ViewerDownload Hi-res image Download (PPT)Knock Down of TDAG51 Expression in NWTb3 Cells—To examine the role of TDAG51 in the functional effects of IGF-I, we generated clones expressing TDAG51 siRNA to knock down TDAG51 expression in NWTb3 cells. The two different TDAG51 siRNA expression vectors, psi21 and psi37, were transfected into NWTb3 cells as described under "Experimental Procedures". Two clones were selected and designated as si21-3 and si37-57. In addition, one of the control clones was cotransfected with pSilencer vector and the hygromycin-resistant plasmid (pcDNA3.1-hygro), which was designated as siHygro1. These hygromicin-resistant clones were analyzed by PCR to confirm that the siRNA expression vectors were expressed in these clones. As shown in Fig. 4A, the selected clones expressed the siRNA construct. We then performed Western blot analysis using an anti-TDAG51 antibody to evaluate protein expression levels in these clones. Because TDAG51 protein is expressed at very low levels in the basal state (in complete culture medium), the cells were stimulated with 50 nm IGF-I for 6 h to induce TDAG51 expression. It can be seen in Fig. 4B that IGF-I robustly induced TDAG51 expression in the parental NWTb3 cells and siHygro1 cells. However, TDAG51 expression was 75-80% lower in si21-3 and si37-57 cells (Fig. 4B), indicating that the siRNA constructs effectively reduced TDAG51 levels. Expression of si21-3 and si37-57 had no effect on actin or IGF-IR expression levels (Fig. 4B).Fig. 4Knock down of TDAG51 expression by siRNA in NWTb3 cells. NWTb3 cells were stably cotransfected with TDAG51 siRNA expression vectors (psi21 or psi37), pSilencer, or pcDNA3.1-hygro, as described under "Experimental Procedures." A, PCR analysis to confirm the expression of siRNA-expressing vectors. Genomic DNA was isolated and analyzed by PCR, as described under "Experimental Procedures." psi21, psi37, and pSilencer served as positive controls. As a negative control, we used genomic DNA derived from NWTb3 cells. B, TDAG51 expression in parental NWTb3 cells and cells expressing siHydro1 or TDAG51 siRNA. Each cell line was subjected to serum starvation and then incubated in the presence or absence of IGF-I (50 nm) for 6 h and homogenized in lysis buffer. The resulting cell lysates were subjected to SDS-PAGE and immunoblotting with an anti-TDAG51 antibody or an antibody directed against the IGF-IR β-subunit. After autoradiography and quantification, membranes were stripped and reprobed with an antibody to actin as the internal control.View Large Image Figure ViewerDownload Hi-res image Download (PPT)TDAG51 Regulates the Inhibitory Effects of IGF-I on Apoptosis Induced by Serum Starvation in NWTb3 Cells—Previous studies have shown that TDAG51 is associated with enhanced apoptosis in several cell lines (8Park C.G. Lee S.Y. Kandala G. Choi Y. Immunity. 1996; 4: 583-591Abstract Full Text Full Text PDF PubMed Scopus (162) Google Scholar, 9Gomes I. Xiong W. Miki T. Rosner M.R. J. Neurochem. 1999; 73: 612-622Crossref PubMed Scopus (57) Google Scholar, 10Neef R. Kuske M.A. Prols E. Johnson J.P. Cancer Res. 2002; 62: 5920-5929PubMed Google Scholar, 25Hossain G.S. van Thienen J.V. Werstuck G.H. Zhou J. Sood S.K. Dickhout J.G. de Koning A.B. Tang D. Wu D. Falk E. Poddar R. Jacobsen D.W. Zhang K. Kaufman R.J. Austin R.C. J. Biol. Chem. 2003; 278: 30317-30327Abstract Full Text Full Text PDF PubMed Scopus (174) Google Scholar). However, in NWTb3 cells, IGF-I (which has an anti-apoptotic function) induced TDAG51 expression. To determine how TDAG51 affects IGF-I function, control cell lines (parental NWTb3 and siHygro1) and clones expressing TDAG51 siRNA (si21-3 and si37-57) were incubated in serum-free medium in the presence or absence of 50 nm IGF-I for 48 h. The cells were then evaluated for apoptosis by Annexin V and 7-AAD staining (Fig. 5A). In control cell lines, the number of apoptotic cells (Annexin V-positive and 7-AAD-negative) represented less than 3% of the population in the basal state (in complete culture medium containing 10% FBS). Apoptotic cells made up about 40% of the population in cells that were subjected to serum starvation for 48 h. In the presence of IGF-I, the population of apoptotic cells was about 15% in control cell lines, reflecting a 60% reduction as compared with cells subjected to serum starvation in the absence of IGF-I. On the other hand, si21-3 and si37-57 cells exhibit
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