Get 20M+ Full-Text Papers For Less Than $1.50/day. Start a 7-Day Trial for You or Your Team.

Learn More →

Transcriptional Effects of Chronic Akt Activation in the Heart

Transcriptional Effects of Chronic Akt Activation in the Heart THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 25, Issue of June 21, pp. 22528 –22533, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Received for publication, February 12, 2002, and in revised form, April 10, 2002 Published, JBC Papers in Press, April 15, 2002, DOI 10.1074/jbc.M201462200 Stuart A. Cook, Takashi Matsui, Ling Li, and Anthony Rosenzweig‡ From the Program in Cardiovascular Gene Therapy, Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02129 Akt activation reduces cardiomyocyte death and in- nase-3, and Bcl-2) identified in other cell types appear to be either expressed at very low levels or not phosphorylated by duces cardiac hypertrophy. To help identify effector mechanisms, gene expression profiles in hearts from Akt in cardiomyocytes (5, 6). These data suggest that addi- transgenic mice with cardiac-specific expression of ac- tional Akt-dependent phosphorylation, translation, and/or tivated Akt (myr-Akt) were compared with littermate transcription events may be required for Akt-mediated cyto- controls. 40 genes were identified as differentially ex- protection in the heart. pressed. Quantitative reverse transcription-PCR con- Translational effects of Akt involve the phosphorylation and firmed qualitative results of transcript profiling for 9 of activation of the mammalian target of rapamycin (mTOR) that 10 genes examined, however, there were notable quan- in turn phosphorylates 4E-BP1 and p70S6 kinase (7). The net titative discrepancies between the quantitative reverse effect of these phosphorylation events is enhanced translation transcription-PCR and microarray data sets. Interest- of specific mRNA subset(s), which is bound by the initiation ingly Akt induced significant up-regulation of insulin- factor eIF-4F and/or the ribosomal S6 subunit. In contrast, the like growth factor-binding protein-5 (IGFBP-5), which transcriptional effects of Akt are less well defined, although the could contribute to its anti-apoptotic effects in the importance of these events may be greater than initially real- heart. In addition, Akt-mediated down-regulation of ized (8, 9). Akt-regulated gene transcription has been described co- peroxisome proliferator-activated receptor (PPAR) for Glut-1 (10), vascular endothelial growth factor (11), and may shift myocytes to- activator-1 (PGC-1) and PPAR- Bcl-2 (12), and a number of Akt-regulated transcription factors ward glycolytic metabolism shown to preserve cardio- have been identified. Akt directly phosphorylates Forkhead box myocyte function and survival during transient ische- transcription factors, class O (FOXOs) (13–15) and may also mia. IGFBP-5 transcripts also increased after adenovi- regulate, through direct and/or indirect mechanisms, AP-1, ral gene transfer of myr-Akt to cultured cardiomyocytes, cAMP-response element-binding protein, and NF-B (16 –19). suggesting that this represents a direct effect of Akt To examine the transcriptional effects of Akt in the heart we activation. In contrast, substantial induction of growth differentiation factor-8 (GDF-8), a highly conserved in- analyzed the changes in global gene expression in transgenic hibitor of skeletal muscle growth, was observed in trans- mice with cardiac-specific expression of myr-Akt using DNA genic hearts but not after acute Akt activation in vitro, microarrays. This approach enabled the quantitation of the suggesting that GDF-8 induction may represent a sec- effects of Akt activation on 11,000 genes. Results of interest ondary effect perhaps related to the cardiac hypertro- were validated by quantitative RT-PCR (QRT-PCR). Here we phy seen in these mice. Thus, microarray analysis re- identify genes differentially regulated by chronic Akt activa- veals previously unappreciated Akt regulation of genes tion in the heart and demonstrate that modulated transcripts that could contribute to the effects of Akt on cardio- represent a combination of primary and secondary effects. The myocyte survival, metabolism, and growth. importance of confirming microarray results of interest using additional, complimentary techniques is discussed. EXPERIMENTAL PROCEDURES The serine-threonine kinase Akt (or protein kinase B) has well documented anti-apoptotic effects in many systems (1–3). Mice—Generation and phenotypic characterization of myr-Akt mice is described elsewhere in detail (20). In brief, the cDNA encoding We have shown that expression of a constitutively active mu- hemagglutinin-tagged Akt with a src myristoylation (myr) signal (kind- tant of Akt (myr-Akt) is sufficient to block apoptosis in hypoxic ly provided by Dr. Thomas F. Franke, Columbia University) was sub- neonatal rat cardiomyocytes in vitro (4) and in vivo prevents cloned downstream of the 5.5-kb murine -myosin heavy chain pro- cardiac injury while preserving heart function during ischemi- moter (generously provided by Dr. Jeffrey Robbins, Division of a-reperfusion injury (5). The downstream targets of Akt that Molecular Cardiovascular Biology, Cincinnati Children’s Hospital Re- mediate cell survival in the heart remain poorly characterized. search Foundation) and used to generate transgenic mice through oo- cyte injection. Positive founders were identified by Southern blotting Indeed some Akt substrates (e.g. Bad, glycogen synthase ki- and bred to wild-type C57BL6 mice for six generations. Two transgenic (TG) lines were maintained; the 20 line exhibited X-linked inheritance, * This work was supported in part by National Institutes of Health whereas the 564 line exhibited autosomal inheritance. TG-positive F3 Grants HL-59521 and HL-61557 (to A. R.) and HL-04250 (to T. M.) and mice were used for studies and compared with TG-negative littermates. a grant from the Wellcome Trust (International Prize Traveling Fel- lowship (to S. A. C.)). The costs of publication of this article were de- frayed in part by the payment of page charges. This article must The abbreviations used are: mTOR, mammalian target of rapamy- therefore be hereby marked “advertisement” in accordance with 18 cin; FOXO, Forkhead box transcription factor, class O; QRT-PCR, quan- U.S.C. Section 1734 solely to indicate this fact. titative reverse transcription-PCR; myr, myristoylated; TG, transgenic; ‡ An established investigator of the American Heart Association. To AvDiff, average difference; Ad, adenoviral vector; EGFP, enhanced whom correspondence should be addressed: Program in Cardiovascular green fluorescent protein; NRVM, neonatal rat ventricular cardiomyo- Gene Therapy, Cardiovascular Research Center, Massachusetts Gen- cyte; MLC1F/3F, myosin alkali light chain 1 fast/3 fast; OTT, ovary eral Hospital-East, 114 16th St., Rm. 2600, Charlestown, MA 02129- testis transcribed; IGF, insulin-like growth factor; IGFBP, IGF-binding 2060. Tel.: 617-726-8286; Fax: 617-726-5806; E-mail: Rosenzweig@ protein; PPAR, peroxisome proliferator-activated receptor; PGC-1, helix.mgh.harvard.edu. PPAR- coactivator-1; GDF-8, growth differentiation factor-8. 22528 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Transcriptional Effects of Chronic Akt Activation in Heart 22529 Both lines express myr-Akt specifically in the heart at levels 5–7-fold fetal bovine serum. Cells were subsequently serum-starved for 24 h higher than the endogenous molecule and exhibit a substantial increase prior to RNA extraction. RNA was extracted, purified, and quantified as in Akt activation as measured by both in vitro kinase assays and in vivo described above. phosphorylation of known substrates (20). Immunoblotting—Hearts from littermate control and myr-Akt-ex- Preparation of cRNA for Microarray Analysis—Total RNA was ex- pressing mice were removed from deeply anesthetized animals, snap tracted from F3, 6-week-old, 20 line male mouse hearts using TRIzol frozen, and crushed under liquid nitrogen before tissue was homoge- M Tris-HCl (pH7.6), 150 mM NaCl, 1% (Invitrogen) according to the manufacturer’s recommendations. RNA nized in cold lysis buffer (20 m Triton X-100, 2 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM was resuspended in diethyl pyrocarbonate-treated H O and further dithiothreitol, 1 mM sodium orthovanadate, 1 g/ml leupeptin, 1 g/ml purified using the Qiagen (Chatsworth, CA) RNeasy total RNA isolation aprotinin). Proteins from NRVMs were extracted by scraping cells di- kit according to the manufacturer’s instructions. RNA was quantified, rectly into cold lysis buffer as described previously (4). Protein concen- and samples (n  2–5 hearts) were pooled such that pooled RNA tration was measured by the Bradford method (Bio-Rad). Proteins (30 represented equal amounts (10 g) of RNA from TG-positive or TG- g) were separated by SDS-PAGE on 12% separation gels and trans- negative mice within the litter. This was repeated in three independent ferred to nitrocellulose membranes (Schleicher & Schuell) by semidry experiments. Pooled samples (10 g) were used to generate cDNA using transfer. Blots were incubated with anti-Akt (1:1000, Cell Signaling) the Superscript Choice system (Invitrogen) according to the Affymetrix overnight at 4 °C and subsequently incubated with horseradish perox- protocol (Affymetrix, Santa Clara, CA). Resulting cDNA was used to idase-conjugated secondary antibody (1:5000, Dako). Immunoreactive generate biotin-labeled cRNA using the ENZO Bioarray High Yield bands were detected by enhanced chemiluminescence (Cell Signaling). transcript labeling kit (Affymetrix). cRNA (20 g) was fragmented in Statistics—Data are represented as mean  S.E. Data were com- fragmentation buffer (40 mM Tris (pH 8.1), 100 mM potassium acetate, pared by two-tailed Student’s t test. The null hypothesis was rejected 30 mM magnesium acetate) for 35 min at 94 °C. The quality of the cRNA for p  0.05. was checked by hybridization to Test2 arrays (Affymetrix) according to the manufacturer’s instructions. Subsequently samples were hybrid- RESULTS ized to Affymetrix mU74A microarrays, and bound sequences were identified by staining and scanning according to Affymetrix protocols. Effects of myr-Akt Expression on Gene Expression in the Analysis of Microarray Data—To enable comparison between exper- Heart—To identify genes differentially regulated by Akt in the iments expression data were globally scaled to an average intensity of TM heart we examined the gene expression profiles of mice with 1500 using the Affymetrix Microarray Suite software. A minimum cardiac-specific expression of myr-Akt (20 line) compared with value of 150 was assigned to all average differences (AvDiffs) with an intensity measurement below 150. Two parameters, the AvDiff and the TG-negative littermate controls. The experiment was repeated absolute call (present or absent), extracted from the Affymetrix data three times to reduce erroneous data that can arise when files, were used in the data analysis, which was performed using Gene- pooled RNA alone is used as a substitute for experimental TM spring (Silicon Genetics, CA). Results were sorted using a combina- replication (25). Genes of interest were identified using the tion of high and low stringency filtering criteria. High stringency filter- described filtering protocols and examined for statistically sig- ing required that a gene should have an absolute call of present in six nificant differences in expression. These analyses revealed that of six samples with a mean -fold change of 1.6. Low stringency filtering required that the gene be called present in two of the three expression of myr-Akt in the heart resulted in the differential replicates in the more highly expressing group with a mean AvDiff of regulation of 40 (21 up-regulated and 19 down-regulated) of the 750 and mean -fold change of 2. Mean -fold changes between groups 11,000 genes examined (Tables I and II). were calculated from the mean AvDiffs. Data passing these criteria It is surprising to observe that the two genes with the great- were combined and subjected to statistical analysis. est -fold changes in expression are not usually expressed in the QRT-PCR Analysis—Total RNA was isolated and purified from the heart. Myosin alkali light chain 1 fast/3 fast (MLC1F/3F, up- hearts of F3, 6-week-old male mice from the 20 and 564 transgenic lines as described above. Following purification RNA was quantified in trip- regulated 11.8-fold) is predominantly expressed in skeletal licate using Ribogreen (Molecular Probes, Eugene, OR) according to the muscle (26) and the ovary testis transcribed (OTT, up-regu- manufacturer’s instructions. RNA (5 g) was treated (10 min at 20 °C) lated 11.1-fold) gene is usually only expressed in the ovary or with amplification grade DNase 1 (Invitrogen) following which the the testis (27). Induction of insulin-like growth factor-binding DNase 1 was heat-inactivated (5 min at 75 °C). QRT-PCR was per- protein-5 (IGFBP-5) by insulin-like growth factor-I (IGF-I) via formed in duplicate using the Brilliant One-Step QRT-PCR kit (Strat- phosphatidylinositol 3-kinase and mTOR has been observed agene, La Jolla, CA) containing SYBR Green I (1:30,000, Sigma), for- ward and reverse primers (50 nM each), and sample RNA (90 ng). previously (28), although a direct connection to Akt has not Primers were designed to be compatible with a single QRT-PCR ther- been reported. Some genes of related function were coordi- mal profile (48 °C for 30 min, 95 °C for 10 min, and 40 cycles of 95 °C for nately regulated by chronic Akt expression. For instance, the 30 s and 60 °C for 1 min) such that multiple transcripts could be potent inhibitor of angiogenesis pigment epithelium-derived analyzed simultaneously. Accumulation of PCR product was monitored factor was up-regulated 2.6-fold, while the angiogenic factor in real time (Mx4000, Stratagene), and the crossing threshold (Ct) was vascular endothelial growth factor was down-regulated 1.8- determined using the Mx4000 software. For each set of primers, a no template control and a no reverse amplification control were included. fold. In addition, transcripts for peroxisome proliferator-acti- Postamplification dissociation curves were performed to verify the pres- vated receptor  (PPAR-) and peroxisome proliferator-acti- ence of a single amplification product in the absence of DNA contami- vated receptor  coactivator-1 (PGC-1), both involved in fatty nation. -Fold changes in gene expression were determined using the acid metabolism, were down-regulated. Ct method with normalization to total RNA (21, 22). Validation of Microarray Data for myr-Akt-expressing Mice Adenoviral Vectors (Ads)—AdEGFP-gal contains cytomegalovirus- by QRT-PCR—The differential expression of six up-regulated driven expression cassettes for -galactosidase and enhanced green fluorescent protein (EGFP) (5). AdAkt(AA) utilizes a similar viral back- and four down-regulated genes, identified by microarray anal- bone but encodes a dominant-negative Akt mutant and was kindly ysis, were validated by QRT-PCR. Relative transcript levels provided by Dr. Wataru Ogawa, Kobe University, Japan (23). Admyr- were determined in F3, 20 line TG-positive males compared Akt and AdEGFP mediate expression of hemagglutinin-tagged consti- with TG-negative male littermate controls (Fig. 1). QRT-PCR tutively active Akt or EGFP, respectively, and have been described analysis confirmed 7 of the 10 genes were statistically differ- previously (5). Ads were amplified in 293 cells, the particle count was entially regulated (p  0.05) in the 20 line. Cardiac ankyrin estimated from A , and the titer was determined by plaque assay. Wild-type adenovirus contamination was excluded by the absence of repeat protein, pigment epithelium-derived factor, and IGF-II, PCR-detectable early region 1 (E1) sequences. although differentially regulated in accordance with microar- In Vitro Studies of myr-Akt Expression—Primary cultures of neona- ray data, did not achieve statistical significance. Cardiac tal rat ventricular cardiomyocytes (NRVMs) were prepared from the ankyrin repeat protein and pigment epithelium-derived factor cardiac ventricles of Sprague-Dawley neonates as described previously were subsequently confirmed as differentially regulated (p (5). To study the effects of transient transgene expression, myocytes 0.05) in the 564 line. Although the -fold change of some genes were infected with adenoviral vectors at a multiplicity of infection of 100 for 24 h in Dulbecco’s modified Eagle’s medium containing 10% (IGFBP-5, pigment epithelium-derived factor, PGC-1, PPAR, 22530 Transcriptional Effects of Chronic Akt Activation in Heart TABLE I Genes significantly up-regulated in myr-Akt-expressing mice Genes identified as up-regulated by microarrays were filtered and analyzed as described. SNAP, soluble N-ethylmaleimide-sensitive factor attachment protein; EST, expressed sequence tag. Gene name -Fold change p GenBank™ MLC1F/MLC3F 11.8 0.01 X12973 Ovary testis transcribed 11.1 0.01 X96603 Insulin-like growth factor-binding protein-5 5.4 0.05 L12447 Growth differentiation factor-8 5.1 0.01 U84005 FXYD ion transport regulator 5 3.6 0.05 U72680 Procollagen, type VIII, 1 2.8 0.05 X66976 Lysozyme P 2.8 0.01 X51547 Golgi SNAP receptor complex member 2 2.7 0.05 AI847904 Pigment epithelium-derived factor 2.6 0.05 AF036164 Cardiac ankyrin repeat protein 2.5 0.01 AF041847 Receptor activity modifying protein 1 2.1 0.05 AJ250489 Complement component 1qc 2.1 0.01 X66295 Peroxisomal biogenesis factor 11a 2.0 0.05 AF093669 Odorant-binding protein Ib 2.0 0.05 AW046850 Ia-associated invariant chain 1.9 0.05 X00496 Heterogeneous nuclear ribonucleoprotein L 1.6 0.01 AB009392 Procollagen C-proteinase enhancer protein 1.6 0.05 X57337 4 ESTs (3.0–1.8) 0.05 TABLE II Genes significantly down-regulated in myr-Akt-expressing mice Genes identified as down-regulated by microarrays were filtered and analyzed as described. EST, expressed sequence tag. Gene name -Fold change p GenBank™ Aryl-hydrocarbon receptor-interacting protein 5.0 0.05 AW227620 Matrin 3 3.0 0.01 AB009275 PGC-1 2.9 0.01 AF049330 Short stature homeobox 2 2.4 0.05 U66918 Homeodomain-interacting protein kinase 3 2.3 0.05 AF077660 Esterase 1 2.2 0.02 AW226939 Cd27-binding protein (SIVA) 2.1 0.05 AF033115 Insulin-like growth factor II 2.1 0.05 X71922 Vascular endothelial growth factor 1.8 0.05 M95200 PPAR- 1.7 0.05 X57638 Methylmalonyl-coenzyme A mutase 1.6 0.05 X51941 8 ESTs (1.8–3.7) 0.05 FIG.1. Comparison of gene expression of sequences identified as differentially regulated by microarray analysis in two myr-Akt- expressing lines. 10 genes identified as differentially regulated in 20 line transgenic hearts by microarray analysis were examined in two myr-Akt-expressing lines (20 line and 564 line) by QRT-PCR using gene-specific primers. Amplified products were detected in real time using SYBR Green I, and product specificity was confirmed by postamplification dissociation curve analysis. Gene expression levels in TG20 and TG564 transgenic hearts were determined relative to littermate controls (n  3– 4 in both groups). A, up-regulated genes: relative expression levels of six up-regulated genes in the 20 line and 564 line myr-Akt-expressing mice. Data are expressed as mean  S.E. (*, p  0.05; **, p  0.01). B, down-regulated genes: relative expression levels of four down-regulated genes in the 20 line and 564 line myr-Akt-expressing mice. VEGF, vascular endothelial growth factor; PEDF, pigment epithelium-derived factor; CARP, cardiac ankyrin repeat protein. and vascular endothelial growth factor), as determined by croarray analysis compared with 675-fold by QRT-PCR (Table QRT-PCR analysis, correlated with the -fold change reported I and Figs. 1 and 2). The second major discrepancy was seen in by microarray analysis there were three major discrepancies. the expression levels of growth differentiation factor-8 (GDF- The greatest discrepancy was observed in the expression levels 8), which was reported as 5.1-fold up-regulated in TG20-posi- of OTT, which was reported as 11.1-fold up-regulated by mi- tive hearts by microarray analysis compared with 18.4-fold Transcriptional Effects of Chronic Akt Activation in Heart 22531 FIG.2. Amplification curves and postamplification dissociation curves for OTT in 20 line mice. Total RNA was prepared from 20 line TG and littermate (LM) controls and subjected to QRT-PCR analysis of OTT mRNA levels using gene-specific primers and postamplification melt curve analysis. A no template control (NTC) and a no amplification control (NAC) were included to confirm accumulation of a single PCR product of the predicted melting temperature in the absence of DNA contamination. A, amplification: amplified product was detected after an average of 18.7 cycles of PCR in TG hearts compared with an average of 28.1 cycles in littermate control hearts (n  3 in both groups). Accumulation of nonspecific product was observed in the no template control after 33 cycles. No amplification was observed in the no amplification control confirming the absence of DNA contamination. B, melting point analysis: the first derivative of the postamplification dissociation curve demonstrates that the accumulated product has a single melting point in accordance with that predicted for the specific OTT amplicon. Minimal nonspecific primer-dimer was observed in the no template control, and no DNA-derived product was observed in the no amplification control. up-regulated by QRT-PCR. These discrepancies may be ex- plained, in part, by the greater dynamic range afforded by QRT-PCR analysis. However, this explanation cannot account for the difference between microarray and QRT-PCR data for MLC1F/3F expression. An 11.8-fold (p  0.01) up-regulation of MLC1F/3F was recorded by microarray analysis compared with a 1.7-fold (p  0.05) up-regulation as determined by QRT- PCR. The relative expression of MLC1F/3F was further exam- ined by Northern blot analysis, which revealed a modest in- crease in MLC1F/3F mRNA levels in TG-positive hearts in FIG.3. Transient expression of myr-Akt increases mRNA en- accordance with the QRT-PCR data and in deference to the coding IGFBP-5 but not that of GDF-8. NRVMs were infected with microarray data (data not shown). AdEGFP, Admyr-Akt, or AdAkt(AA) (multiplicity of infection  100 Comparison of Differential Gene Expression between Two for all), and total RNA or protein was extracted after 24 h in serum-free medium. A, expression of IGFBP-5 and GDF-8 mRNA: relative expres- myr-Akt TG Lines—To control for differences in transgene in- sion levels of IGFBP-5 and GDF-8 were determined by QRT-PCR using sertion, expression, and activity, we determined the relative gene-specific primers. Admyr-Akt increased the expression of IGFBP-5 expression of the 10 genes examined by QRT-PCR in the 20 line by 7.2-fold relative to AdEGFP, whereas AdAkt(AA) did not. In con- in a second myr-Akt-expressing line, the 564 line (Fig. 1). For trast, Admyr-Akt had no effect on expression levels of GDF-8. Data are expressed as mean  S.E. (**, p  0.01; n  3 in all groups). B, all genes except OTT, the pattern of differential expression immunoblots of myr-Akt expression in vivo and in vitro: the expression observed in TG20 mice was confirmed in TG564 mice, although levels of myr-Akt and endogenous Akt were determined to validate the the -fold change in expression was significantly greater in the comparison between in vivo and in vitro QRT-PCR data. Proteins (30 564 line for GDF-8 and IGFBP-5 (64.9 versus 18.4, p  0.01 and g) from hearts or cultured NRVMs were separated by SDS-PAGE, and Akt expression was determined by immunoblotting. Top panel, 20 line 6.0 versus 3.8, p  0.05, respectively; Fig. 1A). Although OTT littermate controls (lanes 1 and 2) and TG positives (lanes 3 and 4). mRNA was detected in the TG564 hearts, there was no differ- Middle panel, 564 line littermate controls (lanes 1 and 2) and TG ence in the low level of expression between TG-positive and positives (lanes 3 and 4). Bottom panel, uninfected NRVMs (lanes 1 and -negative littermates. 2) and NRVMs infected with Admyr-Akt (multiplicity of infection Effects of Transient myr-Akt Expression on IGFBP-5 and 100) for 24 h (lanes 3 and 4). GDF-8 Transcript Levels in Vitro—We next examined whether IGFBP-5 and/or GDF-8 were directly regulated by acute Akt Admyr-Akt did not alter the expression level of GDF-8 at 24 h activation in cardiomyocytes using an in vitro system (4). and had no effect on GDF-8 expression at either 48 or 72 h NRVMs were infected with AdEGFP, Admyr-Akt, or domi- (data not shown). nant-negative AdAkt(AA). AdAkt(AA) served as a full-length DISCUSSION control for the Akt molecule, including the pleckstrin homology domain but lacking catalytic activity. The effects of these con- Akt protects the heart from ischemia-reperfusion injury (5, structs on IGFBP-5 and GDF-8 gene expression were deter- 29), although it does not appear to phosphorylate many of its mined by QRT-PCR (Fig. 3A). Expression of Admyr-Akt, at potential downstream targets, including Bad, when expressed levels comparable to those observed in the TG mice (Fig. 3B), in neonatal or adult cardiomyocytes (5). Thus, the mechanisms significantly up-regulated IGFBP-5 (7.2-fold, p  0.05) com- of Akt cardioprotection remain incompletely defined and may pared with AdAkt(AA). This finding corroborates a previous include transcriptional effects. The recent identification of Akt- study in vascular smooth muscle cells that demonstrated dependent transcripts (e.g. Glut-1, Bcl-2, and Fas ligand) (10 – IGFBP-5 mRNA up-regulation by IGF-I in a phosphatidylinos- 12) and Akt-modulated transcription factors (e.g. FOXOs, AP-1, itol 3-kinase/mTOR-dependent manner (28). In contrast, and cAMP-response element-binding protein) (13–17), which 22532 Transcriptional Effects of Chronic Akt Activation in Heart are expressed in the heart, supports this hypothesis. We char- sequence specificity. The -fold change in expression of GDF-8 in acterized the transcriptional effects of myr-Akt expression in TG20 hearts, compared with littermate controls, was reported the heart using DNA microarrays. as 5.1-fold up-regulated by microarray analysis. In contrast, It has been suggested that DNA microarray experiments analysis of GDF-8 expression in the 20 line by QRT-PCR, likely should be repeated with at least three replicates (25) and that a more accurate means of quantifying mRNA levels, revealed the resulting data sets should be filtered and validated to that GDF-8 was up-regulated by 18.4-fold. This underestima- minimize erroneous data. Indeed, as much as one-third of the tion of -fold change was even greater for OTT, which was found variation seen during an experimental comparison may be to be 11.1-fold up-regulated by microarray analysis compared attributable to variations intrinsic to the arrays themselves with 675-fold by QRT-PCR (Table I and Figs. 1 and 2). The (30). However, data filters should be used with caution as they problem of false positive results reported by microarray anal- can increase the number of false negative results. Thus ysis was illustrated by the MLC1F/3F data, reported as 11.8- changes in important, low copy transcripts, which are excluded fold up-regulated by microarray analysis compared with 1.7- from analysis by virtue of their low AvDiffs and/or their in- fold (20 line) and 1.4-fold (564 line) by QRT-PCR (Table I and creased propensity to be called “absent,” may be missed. We Fig. 1). This false positive result could reflect an error in the observed significant changes in the expression of 40 (0.4%) of sequences on the microarray, the occurrence of which was the genes examined in myr-Akt-expressing hearts (Tables I dramatically demonstrated when up to one-third of the se- and II). Of note, the two transcripts with the greatest -fold quences on one set of mouse arrays were found to be wrong (37). changes, OTT and GDF-8, were in the group of genes identified Other possibilities for this type of error include cross-hybrid- using the “low stringency” filter. This finding illustrates how ization by splice variants, related genes, and/or pseudogenes. potentially important data may be missed if too stringent a The Akt/mTOR pathway has been identified as the crucial filter is applied to microarray data sets. regulator of skeletal muscle and pancreatic islet cell hypertro- We have demonstrated that Akt activation increases the phy in vivo (38, 39). In both our myr-Akt-expressing mouse transcription of IGFBP-5 in the heart. IGFBP-5 may have lines cardiac hypertrophy, with no evidence of decompensation, direct and/or indirect anti-apoptotic activity (31–34). There- was observed at 6 weeks (20). Akt therefore promotes both fore, IGFBP-5 up-regulation, in an Akt-dependent manner, skeletal and cardiac muscle hypertrophy. As Akt promotes may be of particular importance to the cardioprotective effects cardiac hypertrophy, we hypothesize that the observed up- of Akt. In the light of previous studies, Akt-dependent IGFBP-5 regulation of GDF-8, a negative regulator of muscle growth, up-regulation in the heart is likely to be mediated through acts as part of a negative feedback loop limiting heart size. The mTOR (28). It is therefore interesting to note that rapamycin, phenomenon of negative feedback and activation of adaptive an mTOR inhibitor, can dramatically attenuate the protective mechanisms is recognized but infrequently described in trans- effects of insulin, which activates Akt, in the heart (29). In this genic and knockout mice (40, 41). GDF-8, also termed myosta- study, we have also shown that Akt down-regulates PGC-1 and tin, is highly conserved across species, and although first char- PPAR- in the heart. This may shift cardiomyocyte metabolism acterized in skeletal muscle (42, 43) it has also been identified away from fatty acid metabolism in favor of glycolysis, which in the heart (44). The hypothesis that GDF-8 up-regulation is a has been shown to protect cardiomyocytes during transient secondary event is supported by our in vitro experiments where ischemia (35, 36). expression of myr-Akt, at levels similar to those seen in TG Confirmation of microarray data by a previously validated mice (Fig. 3B), resulted in the up-regulation of IGFBP-5 but not and established technique should be performed for a selection GDF-8 (Fig. 3A). It remains unclear whether GDF-8 expression of differentially regulated genes and in particular for genes of is related to myocyte size or organ mass (24). specific interest. Of the 10 genes analyzed by QRT-PCR, nine In summary, these data demonstrate that chronic Akt acti- were confirmed in one or both of the transgenic lines as signif- vation results in the differential regulation of 40 genes in the icantly differentially expressed in keeping with the microarray heart. Several of the observed changes generate intriguing data. However, the degree of differential regulation of OTT, hypotheses regarding the effects of Akt in the heart and possi- GDF-8, and MLC1F/3F determined by QRT-PCR differed ble mechanisms underlying Akt-mediated cardioprotection. Akt- markedly from microarray results (Tables I and II and Fig. 1). dependent up-regulation of the anti-apoptotic molecule OTT mRNA has been described only in the testis and ovary IGFBP-5 may be of particular importance and could contribute (27), and it was initially unclear why this gene should be to the observed cytoprotective effects of Akt in the heart. Sim- up-regulated by Akt activation in the heart. As the inheritance  could shift ilarly Akt down-regulation of PGC-1 and PPAR- in the 20 line is X-linked and OTT is encoded on the X chro- myocytes toward glycolytic metabolism previously shown to mosome (27), we hypothesize that the up-regulation of OTT help preserve cardiomyocyte function and survival during tran- may be an insertional effect of the transgene construct. Con- sient ischemia (35, 36). Chronic Akt activation in the heart was sistent with this hypothesis, OTT was not differentially regu- associated with the differential regulation of a subset of genes lated in the 564 line in which the low level of expression was that are dissimilar to those observed with acute Akt activation similar to that seen in transgene-negative littermates from in other cell types, emphasizing the tissue and temporal spec- both lines and wild-type controls (data not shown). The possi- ificity of changes in transcription profiles (9). In the myr-Akt bility that the discrepancy between the two lines represents an mice, some changes (e.g. IGFBP-5) appear to be direct conse- insertional effect on an autosome in the TG564 mice (for exam- quences of Akt activation and were recapitulated in cardiom- ple in a trans-acting element regulating OTT expression) ap- yocytes in vitro, while other transcripts (e.g. GDF-8) were not pears less likely but has not been formally excluded. As mi- induced by acute Akt activation in vitro and therefore likely croarray characterization of transgenic mice becomes more represent an indirect effect of the transgene. Given the role of common and the murine physical map better characterized, the GDF-8 in limiting skeletal muscle growth, we hypothesize that the dramatic up-regulation of GDF-8 observed in hypertro- hitherto latent frequency of insertional events may become more apparent. phied hearts may represent a negative feedback mechanism. The disparity between microarray and QRT-PCR data for the However, additional studies will be necessary to demonstrate expression levels of GDF-8 and MLC1F/3F highlights two other the functional relevance of the observed alterations in tran- important limitations of microarray data: dynamic range and script levels. Finally, while our transcript profiling and QRT- Transcriptional Effects of Chronic Akt Activation in Heart 22533 20. Matsui, T., Li, L., Wu, J. C., Cook, S. A., Nagoshi, T., Picard, M. H., Liao, R., PCR data were generally concordant, there were some striking and Rosenzweig, A. (2002) J. Biol. Chem. 277, in press discrepancies in the quantitative assessment of mRNA 21. Bustin, S. A. (2000) J. Mol. Endocrinol. 25, 169 –193 changes, underscoring the importance of validation of DNA 22. Pfaffl, M. W. (2001) Nucleic Acids Res. 29, 2002–2007 23. Kitamura, T., Ogawa, W., Sakaue, H., Hino, Y., Kuroda, S., Takata, M., microarray results through additional independent techniques. Matsumoto, M., Maeda, T., Konishi, H., Kikkawa, U., and Kasuga, M. (1998) Mol. Cell. Biol. 18, 3708 –3717 REFERENCES 24. Conlon, I., and Raff, M. (1999) Cell 96, 235–244 1. Dudek, H., Datta, S. R., Franke, T. F., Birnbaum, M. J., Yao, R., Cooper, G. M., 25. Lee, M.-L. T., Kuo, F. C., Whitmore, G. A., and Sklar, J. (2000) Proc. Natl. Segal, R. A., Kaplan, D. R., and Greenberg, M. E. (1997) Science 275, Acad. Sci. U. S. A. 97, 9834 –9839 661– 665 26. Neville, C., Gonzales, D., Houghton, L., McGrew, M. J., and Rosenthal, N. 2. Franke, T. F., Kaplan, D. R., and Cantley, L. C. (1997) Cell 88, 435– 437 (1996) Dev. Genet. 19, 157–162 3. Songyang, Z., Baltimore, D., Cantley, L. C., Kaplan, D. R., and Franke, T. F. 27. Kerr, S. M., Taggart, M. H., Lee, M., and Cooke, H. J. (1996) Hum. Mol. Genet. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11345–11350 5, 1139 –1148 4. Matsui, T., Li, L., del Monte, F., Fukui, Y., Franke, T. F., Hajjar, R. J., and 28. Duan, C., Liimatta, M. B., and Bottum, O. L. (1999) J. Biol. Chem. 274, Rosenzweig, A. (1999) Circulation 100, 2373–2379 37147–37153 5. Matsui, T., Tao, J., del Monte, F., Lee, K. H., Li, L., Picard, M., Force, T. L., 29. Jonassen, A. K., Sack, M. N., Mjos, O. D., and Yellon, D. M. (2001) Circ. Res. Franke, T. F., Hajjar, R. J., and Rosenzweig, A. (2001) Circulation 104, 89, 1191–1198 330 –335 30. Friddle, C. J., Koga, T., Rubin, E. M., and Bristow, J. (2000) Proc. Natl. Acad. 6. Cook, S. A., Sugden, P. H., and Clerk, A. (1999) Circ. Res. 85, 940 –949 Sci. U. S. A. 97, 6745– 6750 7. Proud, C. G., and Denton, R. M. (1997) Biochem. J. 328, 329 –341 31. Clemmons, D. R. (1998) Mol. Cell. Endocrinol. 140, 19 –24 8. Brunet, A., Datta, S. R., and Greenberg, M. E. (2001) Curr. Opin. Neurobiol. 32. Perks, C. M., McCaig, C., and Holly, J. M. (2000) J. Cell. Biochem. 80, 248 –258 11, 297–305 33. Roschier, M., Kuusisto, E., Suuronen, T., Korhonen, P., Kyrylenko, S., and 9. Kuhn, I., Bartholdi, M. F., Salamon, H., Feldman, R. I., Roth, R. A., and Salminen, A. (2001) J. Neurochem. 76, 11–20 Johnson, P. H. (2001) Physiol. Genomics 7, 105–114 34. McCaig, C., Perks, C. M., and Holly, J. M. (2002) J. Cell. Biochem. 84, 784 –794 10. Barthel, A., Okino, S. T., Liao, J., Nakatani, K., Li, J., Whitlock, J. P., Jr., and 35. Apstein, C. S., Gravino, F. N., and Haudenschild, C. C. (1983) Circ. Res. 52, Roth, R. A. (1999) J. Biol. Chem. 274, 20281–20286 515–526 11. Jiang, B.-H., Zheng, J. Z., Aoki, M., and Vogt, P. K. (2000) Proc. Natl. Acad. 36. Malhotra, R., and Brosius, F. C., III (1999) J. Biol. Chem. 274, 12567–12575 Sci. U. S. A. 97, 1749 –1753 37. Knight, J. (2001) Nature 410, 860 – 861 12. Pugazhenthi, S., Nesterova, A., Sable, C., Heidenreich, K. A., Boxer, L. M., 38. Bernal-Mizrachi, E., Wen, W., Stahlhut, S., Welling, C. M., and Permutt, M. A. Heasley, L. E., and Reusch, J. E. (2000) J. Biol. Chem. 275, 10761–10766 (2001) J. Clin. Investig. 108, 1631–1638 13. Biggs, W. H., III, Meisenhelder, J., Hunter, T., Cavenee, W. K., and Arden, 39. Bodine, S. C., Stitt, T. N., Gonzalez, M., Kline, W. O., Stover, G. L., Bauerlein, K. C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 7421–7426 R., Zlotchenko, E., Scrimgeour, A., Lawrence, J. C., Glass, D. J., and 14. Brunet, A., Bonni, A., Zigmond, M. J., Lin, M. Z., Juo, P., Hu, L. S., Anderson, Yancopoulos, G. D. (2001) Nat. Cell Biol. 3, 1014 –1019 M. J., Arden, K. C., Blenis, J., and Greenberg, M. E. (1999) Cell 96, 857– 868 40. Godecke, A., and Schrader, J. (2000) Basic Res. Cardiol. 95, 492– 498 15. Tang, E. D., Nunez, G., Barr, F. G., and Guan, K. L. (1999) J. Biol. Chem. 274, 41. Fishman, G. I. (1998) Circ. Res. 82, 837– 844 16741–16746 42. McPherron, A. C., and Lee, S. J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 16. Kitaura, J., Asai, K., Maeda-Yamamoto, M., Kawakami, Y., Kikkawa, U., and 12457–12461 Kawakami, T. (2000) J. Exp. Med. 192, 729 –740 17. Du, K., and Montminy, M. (1998) J. Biol. Chem. 273, 32377–32379 43. McPherron, A. C., Lawler, A. M., and Lee, S. J. (1997) Nature 387, 83–90 44. Sharma, M., Kambadur, R., Matthews, K. G., Somers, W. G., Devlin, G. P., 18. Ozes, O. N., Mayo, L. D., Gustin, J. A., Pfeffer, S. R., Pfeffer, L. M., and Donner, D. B. (1999) Nature 401, 82– 85 Conaglen, J. V., Fowke, P. J., and Bass, J. J. (1999) J. Cell. Physiol. 19. Romashkova, J. A., and Makarov, S. S. (1999) Nature 401, 86 –90 180, 1–9 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png Journal of Biological Chemistry Unpaywall

Transcriptional Effects of Chronic Akt Activation in the Heart

Cook, Stuart A.; Matsui, Takashi; Li, Ling; Rosenzweig, Anthony
Journal of Biological Chemistry – Jun 1, 2002
Download PDF
6 pages

Loading next page...
 
/lp/unpaywall/transcriptional-effects-of-chronic-akt-activation-in-the-heart-w0nEkyY4XO

References

References for this paper are not available at this time. We will be adding them shortly, thank you for your patience.

Publisher
Unpaywall
ISSN
0021-9258
DOI
10.1074/jbc.m201462200
Publisher site
See Article on Publisher Site

Abstract

THE JOURNAL OF BIOLOGICAL CHEMISTRY Vol. 277, No. 25, Issue of June 21, pp. 22528 –22533, 2002 © 2002 by The American Society for Biochemistry and Molecular Biology, Inc. Printed in U.S.A. Received for publication, February 12, 2002, and in revised form, April 10, 2002 Published, JBC Papers in Press, April 15, 2002, DOI 10.1074/jbc.M201462200 Stuart A. Cook, Takashi Matsui, Ling Li, and Anthony Rosenzweig‡ From the Program in Cardiovascular Gene Therapy, Cardiovascular Research Center, Massachusetts General Hospital, Harvard Medical School, Boston, Massachusetts 02129 Akt activation reduces cardiomyocyte death and in- nase-3, and Bcl-2) identified in other cell types appear to be either expressed at very low levels or not phosphorylated by duces cardiac hypertrophy. To help identify effector mechanisms, gene expression profiles in hearts from Akt in cardiomyocytes (5, 6). These data suggest that addi- transgenic mice with cardiac-specific expression of ac- tional Akt-dependent phosphorylation, translation, and/or tivated Akt (myr-Akt) were compared with littermate transcription events may be required for Akt-mediated cyto- controls. 40 genes were identified as differentially ex- protection in the heart. pressed. Quantitative reverse transcription-PCR con- Translational effects of Akt involve the phosphorylation and firmed qualitative results of transcript profiling for 9 of activation of the mammalian target of rapamycin (mTOR) that 10 genes examined, however, there were notable quan- in turn phosphorylates 4E-BP1 and p70S6 kinase (7). The net titative discrepancies between the quantitative reverse effect of these phosphorylation events is enhanced translation transcription-PCR and microarray data sets. Interest- of specific mRNA subset(s), which is bound by the initiation ingly Akt induced significant up-regulation of insulin- factor eIF-4F and/or the ribosomal S6 subunit. In contrast, the like growth factor-binding protein-5 (IGFBP-5), which transcriptional effects of Akt are less well defined, although the could contribute to its anti-apoptotic effects in the importance of these events may be greater than initially real- heart. In addition, Akt-mediated down-regulation of ized (8, 9). Akt-regulated gene transcription has been described co- peroxisome proliferator-activated receptor (PPAR) for Glut-1 (10), vascular endothelial growth factor (11), and may shift myocytes to- activator-1 (PGC-1) and PPAR- Bcl-2 (12), and a number of Akt-regulated transcription factors ward glycolytic metabolism shown to preserve cardio- have been identified. Akt directly phosphorylates Forkhead box myocyte function and survival during transient ische- transcription factors, class O (FOXOs) (13–15) and may also mia. IGFBP-5 transcripts also increased after adenovi- regulate, through direct and/or indirect mechanisms, AP-1, ral gene transfer of myr-Akt to cultured cardiomyocytes, cAMP-response element-binding protein, and NF-B (16 –19). suggesting that this represents a direct effect of Akt To examine the transcriptional effects of Akt in the heart we activation. In contrast, substantial induction of growth differentiation factor-8 (GDF-8), a highly conserved in- analyzed the changes in global gene expression in transgenic hibitor of skeletal muscle growth, was observed in trans- mice with cardiac-specific expression of myr-Akt using DNA genic hearts but not after acute Akt activation in vitro, microarrays. This approach enabled the quantitation of the suggesting that GDF-8 induction may represent a sec- effects of Akt activation on 11,000 genes. Results of interest ondary effect perhaps related to the cardiac hypertro- were validated by quantitative RT-PCR (QRT-PCR). Here we phy seen in these mice. Thus, microarray analysis re- identify genes differentially regulated by chronic Akt activa- veals previously unappreciated Akt regulation of genes tion in the heart and demonstrate that modulated transcripts that could contribute to the effects of Akt on cardio- represent a combination of primary and secondary effects. The myocyte survival, metabolism, and growth. importance of confirming microarray results of interest using additional, complimentary techniques is discussed. EXPERIMENTAL PROCEDURES The serine-threonine kinase Akt (or protein kinase B) has well documented anti-apoptotic effects in many systems (1–3). Mice—Generation and phenotypic characterization of myr-Akt mice is described elsewhere in detail (20). In brief, the cDNA encoding We have shown that expression of a constitutively active mu- hemagglutinin-tagged Akt with a src myristoylation (myr) signal (kind- tant of Akt (myr-Akt) is sufficient to block apoptosis in hypoxic ly provided by Dr. Thomas F. Franke, Columbia University) was sub- neonatal rat cardiomyocytes in vitro (4) and in vivo prevents cloned downstream of the 5.5-kb murine -myosin heavy chain pro- cardiac injury while preserving heart function during ischemi- moter (generously provided by Dr. Jeffrey Robbins, Division of a-reperfusion injury (5). The downstream targets of Akt that Molecular Cardiovascular Biology, Cincinnati Children’s Hospital Re- mediate cell survival in the heart remain poorly characterized. search Foundation) and used to generate transgenic mice through oo- cyte injection. Positive founders were identified by Southern blotting Indeed some Akt substrates (e.g. Bad, glycogen synthase ki- and bred to wild-type C57BL6 mice for six generations. Two transgenic (TG) lines were maintained; the 20 line exhibited X-linked inheritance, * This work was supported in part by National Institutes of Health whereas the 564 line exhibited autosomal inheritance. TG-positive F3 Grants HL-59521 and HL-61557 (to A. R.) and HL-04250 (to T. M.) and mice were used for studies and compared with TG-negative littermates. a grant from the Wellcome Trust (International Prize Traveling Fel- lowship (to S. A. C.)). The costs of publication of this article were de- frayed in part by the payment of page charges. This article must The abbreviations used are: mTOR, mammalian target of rapamy- therefore be hereby marked “advertisement” in accordance with 18 cin; FOXO, Forkhead box transcription factor, class O; QRT-PCR, quan- U.S.C. Section 1734 solely to indicate this fact. titative reverse transcription-PCR; myr, myristoylated; TG, transgenic; ‡ An established investigator of the American Heart Association. To AvDiff, average difference; Ad, adenoviral vector; EGFP, enhanced whom correspondence should be addressed: Program in Cardiovascular green fluorescent protein; NRVM, neonatal rat ventricular cardiomyo- Gene Therapy, Cardiovascular Research Center, Massachusetts Gen- cyte; MLC1F/3F, myosin alkali light chain 1 fast/3 fast; OTT, ovary eral Hospital-East, 114 16th St., Rm. 2600, Charlestown, MA 02129- testis transcribed; IGF, insulin-like growth factor; IGFBP, IGF-binding 2060. Tel.: 617-726-8286; Fax: 617-726-5806; E-mail: Rosenzweig@ protein; PPAR, peroxisome proliferator-activated receptor; PGC-1, helix.mgh.harvard.edu. PPAR- coactivator-1; GDF-8, growth differentiation factor-8. 22528 This paper is available on line at http://www.jbc.org This is an Open Access article under the CC BY license. Transcriptional Effects of Chronic Akt Activation in Heart 22529 Both lines express myr-Akt specifically in the heart at levels 5–7-fold fetal bovine serum. Cells were subsequently serum-starved for 24 h higher than the endogenous molecule and exhibit a substantial increase prior to RNA extraction. RNA was extracted, purified, and quantified as in Akt activation as measured by both in vitro kinase assays and in vivo described above. phosphorylation of known substrates (20). Immunoblotting—Hearts from littermate control and myr-Akt-ex- Preparation of cRNA for Microarray Analysis—Total RNA was ex- pressing mice were removed from deeply anesthetized animals, snap tracted from F3, 6-week-old, 20 line male mouse hearts using TRIzol frozen, and crushed under liquid nitrogen before tissue was homoge- M Tris-HCl (pH7.6), 150 mM NaCl, 1% (Invitrogen) according to the manufacturer’s recommendations. RNA nized in cold lysis buffer (20 m Triton X-100, 2 mM EGTA, 1 mM phenylmethylsulfonyl fluoride, 1 mM was resuspended in diethyl pyrocarbonate-treated H O and further dithiothreitol, 1 mM sodium orthovanadate, 1 g/ml leupeptin, 1 g/ml purified using the Qiagen (Chatsworth, CA) RNeasy total RNA isolation aprotinin). Proteins from NRVMs were extracted by scraping cells di- kit according to the manufacturer’s instructions. RNA was quantified, rectly into cold lysis buffer as described previously (4). Protein concen- and samples (n  2–5 hearts) were pooled such that pooled RNA tration was measured by the Bradford method (Bio-Rad). Proteins (30 represented equal amounts (10 g) of RNA from TG-positive or TG- g) were separated by SDS-PAGE on 12% separation gels and trans- negative mice within the litter. This was repeated in three independent ferred to nitrocellulose membranes (Schleicher & Schuell) by semidry experiments. Pooled samples (10 g) were used to generate cDNA using transfer. Blots were incubated with anti-Akt (1:1000, Cell Signaling) the Superscript Choice system (Invitrogen) according to the Affymetrix overnight at 4 °C and subsequently incubated with horseradish perox- protocol (Affymetrix, Santa Clara, CA). Resulting cDNA was used to idase-conjugated secondary antibody (1:5000, Dako). Immunoreactive generate biotin-labeled cRNA using the ENZO Bioarray High Yield bands were detected by enhanced chemiluminescence (Cell Signaling). transcript labeling kit (Affymetrix). cRNA (20 g) was fragmented in Statistics—Data are represented as mean  S.E. Data were com- fragmentation buffer (40 mM Tris (pH 8.1), 100 mM potassium acetate, pared by two-tailed Student’s t test. The null hypothesis was rejected 30 mM magnesium acetate) for 35 min at 94 °C. The quality of the cRNA for p  0.05. was checked by hybridization to Test2 arrays (Affymetrix) according to the manufacturer’s instructions. Subsequently samples were hybrid- RESULTS ized to Affymetrix mU74A microarrays, and bound sequences were identified by staining and scanning according to Affymetrix protocols. Effects of myr-Akt Expression on Gene Expression in the Analysis of Microarray Data—To enable comparison between exper- Heart—To identify genes differentially regulated by Akt in the iments expression data were globally scaled to an average intensity of TM heart we examined the gene expression profiles of mice with 1500 using the Affymetrix Microarray Suite software. A minimum cardiac-specific expression of myr-Akt (20 line) compared with value of 150 was assigned to all average differences (AvDiffs) with an intensity measurement below 150. Two parameters, the AvDiff and the TG-negative littermate controls. The experiment was repeated absolute call (present or absent), extracted from the Affymetrix data three times to reduce erroneous data that can arise when files, were used in the data analysis, which was performed using Gene- pooled RNA alone is used as a substitute for experimental TM spring (Silicon Genetics, CA). Results were sorted using a combina- replication (25). Genes of interest were identified using the tion of high and low stringency filtering criteria. High stringency filter- described filtering protocols and examined for statistically sig- ing required that a gene should have an absolute call of present in six nificant differences in expression. These analyses revealed that of six samples with a mean -fold change of 1.6. Low stringency filtering required that the gene be called present in two of the three expression of myr-Akt in the heart resulted in the differential replicates in the more highly expressing group with a mean AvDiff of regulation of 40 (21 up-regulated and 19 down-regulated) of the 750 and mean -fold change of 2. Mean -fold changes between groups 11,000 genes examined (Tables I and II). were calculated from the mean AvDiffs. Data passing these criteria It is surprising to observe that the two genes with the great- were combined and subjected to statistical analysis. est -fold changes in expression are not usually expressed in the QRT-PCR Analysis—Total RNA was isolated and purified from the heart. Myosin alkali light chain 1 fast/3 fast (MLC1F/3F, up- hearts of F3, 6-week-old male mice from the 20 and 564 transgenic lines as described above. Following purification RNA was quantified in trip- regulated 11.8-fold) is predominantly expressed in skeletal licate using Ribogreen (Molecular Probes, Eugene, OR) according to the muscle (26) and the ovary testis transcribed (OTT, up-regu- manufacturer’s instructions. RNA (5 g) was treated (10 min at 20 °C) lated 11.1-fold) gene is usually only expressed in the ovary or with amplification grade DNase 1 (Invitrogen) following which the the testis (27). Induction of insulin-like growth factor-binding DNase 1 was heat-inactivated (5 min at 75 °C). QRT-PCR was per- protein-5 (IGFBP-5) by insulin-like growth factor-I (IGF-I) via formed in duplicate using the Brilliant One-Step QRT-PCR kit (Strat- phosphatidylinositol 3-kinase and mTOR has been observed agene, La Jolla, CA) containing SYBR Green I (1:30,000, Sigma), for- ward and reverse primers (50 nM each), and sample RNA (90 ng). previously (28), although a direct connection to Akt has not Primers were designed to be compatible with a single QRT-PCR ther- been reported. Some genes of related function were coordi- mal profile (48 °C for 30 min, 95 °C for 10 min, and 40 cycles of 95 °C for nately regulated by chronic Akt expression. For instance, the 30 s and 60 °C for 1 min) such that multiple transcripts could be potent inhibitor of angiogenesis pigment epithelium-derived analyzed simultaneously. Accumulation of PCR product was monitored factor was up-regulated 2.6-fold, while the angiogenic factor in real time (Mx4000, Stratagene), and the crossing threshold (Ct) was vascular endothelial growth factor was down-regulated 1.8- determined using the Mx4000 software. For each set of primers, a no template control and a no reverse amplification control were included. fold. In addition, transcripts for peroxisome proliferator-acti- Postamplification dissociation curves were performed to verify the pres- vated receptor  (PPAR-) and peroxisome proliferator-acti- ence of a single amplification product in the absence of DNA contami- vated receptor  coactivator-1 (PGC-1), both involved in fatty nation. -Fold changes in gene expression were determined using the acid metabolism, were down-regulated. Ct method with normalization to total RNA (21, 22). Validation of Microarray Data for myr-Akt-expressing Mice Adenoviral Vectors (Ads)—AdEGFP-gal contains cytomegalovirus- by QRT-PCR—The differential expression of six up-regulated driven expression cassettes for -galactosidase and enhanced green fluorescent protein (EGFP) (5). AdAkt(AA) utilizes a similar viral back- and four down-regulated genes, identified by microarray anal- bone but encodes a dominant-negative Akt mutant and was kindly ysis, were validated by QRT-PCR. Relative transcript levels provided by Dr. Wataru Ogawa, Kobe University, Japan (23). Admyr- were determined in F3, 20 line TG-positive males compared Akt and AdEGFP mediate expression of hemagglutinin-tagged consti- with TG-negative male littermate controls (Fig. 1). QRT-PCR tutively active Akt or EGFP, respectively, and have been described analysis confirmed 7 of the 10 genes were statistically differ- previously (5). Ads were amplified in 293 cells, the particle count was entially regulated (p  0.05) in the 20 line. Cardiac ankyrin estimated from A , and the titer was determined by plaque assay. Wild-type adenovirus contamination was excluded by the absence of repeat protein, pigment epithelium-derived factor, and IGF-II, PCR-detectable early region 1 (E1) sequences. although differentially regulated in accordance with microar- In Vitro Studies of myr-Akt Expression—Primary cultures of neona- ray data, did not achieve statistical significance. Cardiac tal rat ventricular cardiomyocytes (NRVMs) were prepared from the ankyrin repeat protein and pigment epithelium-derived factor cardiac ventricles of Sprague-Dawley neonates as described previously were subsequently confirmed as differentially regulated (p (5). To study the effects of transient transgene expression, myocytes 0.05) in the 564 line. Although the -fold change of some genes were infected with adenoviral vectors at a multiplicity of infection of 100 for 24 h in Dulbecco’s modified Eagle’s medium containing 10% (IGFBP-5, pigment epithelium-derived factor, PGC-1, PPAR, 22530 Transcriptional Effects of Chronic Akt Activation in Heart TABLE I Genes significantly up-regulated in myr-Akt-expressing mice Genes identified as up-regulated by microarrays were filtered and analyzed as described. SNAP, soluble N-ethylmaleimide-sensitive factor attachment protein; EST, expressed sequence tag. Gene name -Fold change p GenBank™ MLC1F/MLC3F 11.8 0.01 X12973 Ovary testis transcribed 11.1 0.01 X96603 Insulin-like growth factor-binding protein-5 5.4 0.05 L12447 Growth differentiation factor-8 5.1 0.01 U84005 FXYD ion transport regulator 5 3.6 0.05 U72680 Procollagen, type VIII, 1 2.8 0.05 X66976 Lysozyme P 2.8 0.01 X51547 Golgi SNAP receptor complex member 2 2.7 0.05 AI847904 Pigment epithelium-derived factor 2.6 0.05 AF036164 Cardiac ankyrin repeat protein 2.5 0.01 AF041847 Receptor activity modifying protein 1 2.1 0.05 AJ250489 Complement component 1qc 2.1 0.01 X66295 Peroxisomal biogenesis factor 11a 2.0 0.05 AF093669 Odorant-binding protein Ib 2.0 0.05 AW046850 Ia-associated invariant chain 1.9 0.05 X00496 Heterogeneous nuclear ribonucleoprotein L 1.6 0.01 AB009392 Procollagen C-proteinase enhancer protein 1.6 0.05 X57337 4 ESTs (3.0–1.8) 0.05 TABLE II Genes significantly down-regulated in myr-Akt-expressing mice Genes identified as down-regulated by microarrays were filtered and analyzed as described. EST, expressed sequence tag. Gene name -Fold change p GenBank™ Aryl-hydrocarbon receptor-interacting protein 5.0 0.05 AW227620 Matrin 3 3.0 0.01 AB009275 PGC-1 2.9 0.01 AF049330 Short stature homeobox 2 2.4 0.05 U66918 Homeodomain-interacting protein kinase 3 2.3 0.05 AF077660 Esterase 1 2.2 0.02 AW226939 Cd27-binding protein (SIVA) 2.1 0.05 AF033115 Insulin-like growth factor II 2.1 0.05 X71922 Vascular endothelial growth factor 1.8 0.05 M95200 PPAR- 1.7 0.05 X57638 Methylmalonyl-coenzyme A mutase 1.6 0.05 X51941 8 ESTs (1.8–3.7) 0.05 FIG.1. Comparison of gene expression of sequences identified as differentially regulated by microarray analysis in two myr-Akt- expressing lines. 10 genes identified as differentially regulated in 20 line transgenic hearts by microarray analysis were examined in two myr-Akt-expressing lines (20 line and 564 line) by QRT-PCR using gene-specific primers. Amplified products were detected in real time using SYBR Green I, and product specificity was confirmed by postamplification dissociation curve analysis. Gene expression levels in TG20 and TG564 transgenic hearts were determined relative to littermate controls (n  3– 4 in both groups). A, up-regulated genes: relative expression levels of six up-regulated genes in the 20 line and 564 line myr-Akt-expressing mice. Data are expressed as mean  S.E. (*, p  0.05; **, p  0.01). B, down-regulated genes: relative expression levels of four down-regulated genes in the 20 line and 564 line myr-Akt-expressing mice. VEGF, vascular endothelial growth factor; PEDF, pigment epithelium-derived factor; CARP, cardiac ankyrin repeat protein. and vascular endothelial growth factor), as determined by croarray analysis compared with 675-fold by QRT-PCR (Table QRT-PCR analysis, correlated with the -fold change reported I and Figs. 1 and 2). The second major discrepancy was seen in by microarray analysis there were three major discrepancies. the expression levels of growth differentiation factor-8 (GDF- The greatest discrepancy was observed in the expression levels 8), which was reported as 5.1-fold up-regulated in TG20-posi- of OTT, which was reported as 11.1-fold up-regulated by mi- tive hearts by microarray analysis compared with 18.4-fold Transcriptional Effects of Chronic Akt Activation in Heart 22531 FIG.2. Amplification curves and postamplification dissociation curves for OTT in 20 line mice. Total RNA was prepared from 20 line TG and littermate (LM) controls and subjected to QRT-PCR analysis of OTT mRNA levels using gene-specific primers and postamplification melt curve analysis. A no template control (NTC) and a no amplification control (NAC) were included to confirm accumulation of a single PCR product of the predicted melting temperature in the absence of DNA contamination. A, amplification: amplified product was detected after an average of 18.7 cycles of PCR in TG hearts compared with an average of 28.1 cycles in littermate control hearts (n  3 in both groups). Accumulation of nonspecific product was observed in the no template control after 33 cycles. No amplification was observed in the no amplification control confirming the absence of DNA contamination. B, melting point analysis: the first derivative of the postamplification dissociation curve demonstrates that the accumulated product has a single melting point in accordance with that predicted for the specific OTT amplicon. Minimal nonspecific primer-dimer was observed in the no template control, and no DNA-derived product was observed in the no amplification control. up-regulated by QRT-PCR. These discrepancies may be ex- plained, in part, by the greater dynamic range afforded by QRT-PCR analysis. However, this explanation cannot account for the difference between microarray and QRT-PCR data for MLC1F/3F expression. An 11.8-fold (p  0.01) up-regulation of MLC1F/3F was recorded by microarray analysis compared with a 1.7-fold (p  0.05) up-regulation as determined by QRT- PCR. The relative expression of MLC1F/3F was further exam- ined by Northern blot analysis, which revealed a modest in- crease in MLC1F/3F mRNA levels in TG-positive hearts in FIG.3. Transient expression of myr-Akt increases mRNA en- accordance with the QRT-PCR data and in deference to the coding IGFBP-5 but not that of GDF-8. NRVMs were infected with microarray data (data not shown). AdEGFP, Admyr-Akt, or AdAkt(AA) (multiplicity of infection  100 Comparison of Differential Gene Expression between Two for all), and total RNA or protein was extracted after 24 h in serum-free medium. A, expression of IGFBP-5 and GDF-8 mRNA: relative expres- myr-Akt TG Lines—To control for differences in transgene in- sion levels of IGFBP-5 and GDF-8 were determined by QRT-PCR using sertion, expression, and activity, we determined the relative gene-specific primers. Admyr-Akt increased the expression of IGFBP-5 expression of the 10 genes examined by QRT-PCR in the 20 line by 7.2-fold relative to AdEGFP, whereas AdAkt(AA) did not. In con- in a second myr-Akt-expressing line, the 564 line (Fig. 1). For trast, Admyr-Akt had no effect on expression levels of GDF-8. Data are expressed as mean  S.E. (**, p  0.01; n  3 in all groups). B, all genes except OTT, the pattern of differential expression immunoblots of myr-Akt expression in vivo and in vitro: the expression observed in TG20 mice was confirmed in TG564 mice, although levels of myr-Akt and endogenous Akt were determined to validate the the -fold change in expression was significantly greater in the comparison between in vivo and in vitro QRT-PCR data. Proteins (30 564 line for GDF-8 and IGFBP-5 (64.9 versus 18.4, p  0.01 and g) from hearts or cultured NRVMs were separated by SDS-PAGE, and Akt expression was determined by immunoblotting. Top panel, 20 line 6.0 versus 3.8, p  0.05, respectively; Fig. 1A). Although OTT littermate controls (lanes 1 and 2) and TG positives (lanes 3 and 4). mRNA was detected in the TG564 hearts, there was no differ- Middle panel, 564 line littermate controls (lanes 1 and 2) and TG ence in the low level of expression between TG-positive and positives (lanes 3 and 4). Bottom panel, uninfected NRVMs (lanes 1 and -negative littermates. 2) and NRVMs infected with Admyr-Akt (multiplicity of infection Effects of Transient myr-Akt Expression on IGFBP-5 and 100) for 24 h (lanes 3 and 4). GDF-8 Transcript Levels in Vitro—We next examined whether IGFBP-5 and/or GDF-8 were directly regulated by acute Akt Admyr-Akt did not alter the expression level of GDF-8 at 24 h activation in cardiomyocytes using an in vitro system (4). and had no effect on GDF-8 expression at either 48 or 72 h NRVMs were infected with AdEGFP, Admyr-Akt, or domi- (data not shown). nant-negative AdAkt(AA). AdAkt(AA) served as a full-length DISCUSSION control for the Akt molecule, including the pleckstrin homology domain but lacking catalytic activity. The effects of these con- Akt protects the heart from ischemia-reperfusion injury (5, structs on IGFBP-5 and GDF-8 gene expression were deter- 29), although it does not appear to phosphorylate many of its mined by QRT-PCR (Fig. 3A). Expression of Admyr-Akt, at potential downstream targets, including Bad, when expressed levels comparable to those observed in the TG mice (Fig. 3B), in neonatal or adult cardiomyocytes (5). Thus, the mechanisms significantly up-regulated IGFBP-5 (7.2-fold, p  0.05) com- of Akt cardioprotection remain incompletely defined and may pared with AdAkt(AA). This finding corroborates a previous include transcriptional effects. The recent identification of Akt- study in vascular smooth muscle cells that demonstrated dependent transcripts (e.g. Glut-1, Bcl-2, and Fas ligand) (10 – IGFBP-5 mRNA up-regulation by IGF-I in a phosphatidylinos- 12) and Akt-modulated transcription factors (e.g. FOXOs, AP-1, itol 3-kinase/mTOR-dependent manner (28). In contrast, and cAMP-response element-binding protein) (13–17), which 22532 Transcriptional Effects of Chronic Akt Activation in Heart are expressed in the heart, supports this hypothesis. We char- sequence specificity. The -fold change in expression of GDF-8 in acterized the transcriptional effects of myr-Akt expression in TG20 hearts, compared with littermate controls, was reported the heart using DNA microarrays. as 5.1-fold up-regulated by microarray analysis. In contrast, It has been suggested that DNA microarray experiments analysis of GDF-8 expression in the 20 line by QRT-PCR, likely should be repeated with at least three replicates (25) and that a more accurate means of quantifying mRNA levels, revealed the resulting data sets should be filtered and validated to that GDF-8 was up-regulated by 18.4-fold. This underestima- minimize erroneous data. Indeed, as much as one-third of the tion of -fold change was even greater for OTT, which was found variation seen during an experimental comparison may be to be 11.1-fold up-regulated by microarray analysis compared attributable to variations intrinsic to the arrays themselves with 675-fold by QRT-PCR (Table I and Figs. 1 and 2). The (30). However, data filters should be used with caution as they problem of false positive results reported by microarray anal- can increase the number of false negative results. Thus ysis was illustrated by the MLC1F/3F data, reported as 11.8- changes in important, low copy transcripts, which are excluded fold up-regulated by microarray analysis compared with 1.7- from analysis by virtue of their low AvDiffs and/or their in- fold (20 line) and 1.4-fold (564 line) by QRT-PCR (Table I and creased propensity to be called “absent,” may be missed. We Fig. 1). This false positive result could reflect an error in the observed significant changes in the expression of 40 (0.4%) of sequences on the microarray, the occurrence of which was the genes examined in myr-Akt-expressing hearts (Tables I dramatically demonstrated when up to one-third of the se- and II). Of note, the two transcripts with the greatest -fold quences on one set of mouse arrays were found to be wrong (37). changes, OTT and GDF-8, were in the group of genes identified Other possibilities for this type of error include cross-hybrid- using the “low stringency” filter. This finding illustrates how ization by splice variants, related genes, and/or pseudogenes. potentially important data may be missed if too stringent a The Akt/mTOR pathway has been identified as the crucial filter is applied to microarray data sets. regulator of skeletal muscle and pancreatic islet cell hypertro- We have demonstrated that Akt activation increases the phy in vivo (38, 39). In both our myr-Akt-expressing mouse transcription of IGFBP-5 in the heart. IGFBP-5 may have lines cardiac hypertrophy, with no evidence of decompensation, direct and/or indirect anti-apoptotic activity (31–34). There- was observed at 6 weeks (20). Akt therefore promotes both fore, IGFBP-5 up-regulation, in an Akt-dependent manner, skeletal and cardiac muscle hypertrophy. As Akt promotes may be of particular importance to the cardioprotective effects cardiac hypertrophy, we hypothesize that the observed up- of Akt. In the light of previous studies, Akt-dependent IGFBP-5 regulation of GDF-8, a negative regulator of muscle growth, up-regulation in the heart is likely to be mediated through acts as part of a negative feedback loop limiting heart size. The mTOR (28). It is therefore interesting to note that rapamycin, phenomenon of negative feedback and activation of adaptive an mTOR inhibitor, can dramatically attenuate the protective mechanisms is recognized but infrequently described in trans- effects of insulin, which activates Akt, in the heart (29). In this genic and knockout mice (40, 41). GDF-8, also termed myosta- study, we have also shown that Akt down-regulates PGC-1 and tin, is highly conserved across species, and although first char- PPAR- in the heart. This may shift cardiomyocyte metabolism acterized in skeletal muscle (42, 43) it has also been identified away from fatty acid metabolism in favor of glycolysis, which in the heart (44). The hypothesis that GDF-8 up-regulation is a has been shown to protect cardiomyocytes during transient secondary event is supported by our in vitro experiments where ischemia (35, 36). expression of myr-Akt, at levels similar to those seen in TG Confirmation of microarray data by a previously validated mice (Fig. 3B), resulted in the up-regulation of IGFBP-5 but not and established technique should be performed for a selection GDF-8 (Fig. 3A). It remains unclear whether GDF-8 expression of differentially regulated genes and in particular for genes of is related to myocyte size or organ mass (24). specific interest. Of the 10 genes analyzed by QRT-PCR, nine In summary, these data demonstrate that chronic Akt acti- were confirmed in one or both of the transgenic lines as signif- vation results in the differential regulation of 40 genes in the icantly differentially expressed in keeping with the microarray heart. Several of the observed changes generate intriguing data. However, the degree of differential regulation of OTT, hypotheses regarding the effects of Akt in the heart and possi- GDF-8, and MLC1F/3F determined by QRT-PCR differed ble mechanisms underlying Akt-mediated cardioprotection. Akt- markedly from microarray results (Tables I and II and Fig. 1). dependent up-regulation of the anti-apoptotic molecule OTT mRNA has been described only in the testis and ovary IGFBP-5 may be of particular importance and could contribute (27), and it was initially unclear why this gene should be to the observed cytoprotective effects of Akt in the heart. Sim- up-regulated by Akt activation in the heart. As the inheritance  could shift ilarly Akt down-regulation of PGC-1 and PPAR- in the 20 line is X-linked and OTT is encoded on the X chro- myocytes toward glycolytic metabolism previously shown to mosome (27), we hypothesize that the up-regulation of OTT help preserve cardiomyocyte function and survival during tran- may be an insertional effect of the transgene construct. Con- sient ischemia (35, 36). Chronic Akt activation in the heart was sistent with this hypothesis, OTT was not differentially regu- associated with the differential regulation of a subset of genes lated in the 564 line in which the low level of expression was that are dissimilar to those observed with acute Akt activation similar to that seen in transgene-negative littermates from in other cell types, emphasizing the tissue and temporal spec- both lines and wild-type controls (data not shown). The possi- ificity of changes in transcription profiles (9). In the myr-Akt bility that the discrepancy between the two lines represents an mice, some changes (e.g. IGFBP-5) appear to be direct conse- insertional effect on an autosome in the TG564 mice (for exam- quences of Akt activation and were recapitulated in cardiom- ple in a trans-acting element regulating OTT expression) ap- yocytes in vitro, while other transcripts (e.g. GDF-8) were not pears less likely but has not been formally excluded. As mi- induced by acute Akt activation in vitro and therefore likely croarray characterization of transgenic mice becomes more represent an indirect effect of the transgene. Given the role of common and the murine physical map better characterized, the GDF-8 in limiting skeletal muscle growth, we hypothesize that the dramatic up-regulation of GDF-8 observed in hypertro- hitherto latent frequency of insertional events may become more apparent. phied hearts may represent a negative feedback mechanism. The disparity between microarray and QRT-PCR data for the However, additional studies will be necessary to demonstrate expression levels of GDF-8 and MLC1F/3F highlights two other the functional relevance of the observed alterations in tran- important limitations of microarray data: dynamic range and script levels. Finally, while our transcript profiling and QRT- Transcriptional Effects of Chronic Akt Activation in Heart 22533 20. Matsui, T., Li, L., Wu, J. C., Cook, S. A., Nagoshi, T., Picard, M. H., Liao, R., PCR data were generally concordant, there were some striking and Rosenzweig, A. (2002) J. Biol. Chem. 277, in press discrepancies in the quantitative assessment of mRNA 21. Bustin, S. A. (2000) J. Mol. Endocrinol. 25, 169 –193 changes, underscoring the importance of validation of DNA 22. Pfaffl, M. W. (2001) Nucleic Acids Res. 29, 2002–2007 23. Kitamura, T., Ogawa, W., Sakaue, H., Hino, Y., Kuroda, S., Takata, M., microarray results through additional independent techniques. Matsumoto, M., Maeda, T., Konishi, H., Kikkawa, U., and Kasuga, M. (1998) Mol. Cell. Biol. 18, 3708 –3717 REFERENCES 24. Conlon, I., and Raff, M. (1999) Cell 96, 235–244 1. Dudek, H., Datta, S. R., Franke, T. F., Birnbaum, M. J., Yao, R., Cooper, G. M., 25. Lee, M.-L. T., Kuo, F. C., Whitmore, G. A., and Sklar, J. (2000) Proc. Natl. Segal, R. A., Kaplan, D. R., and Greenberg, M. E. (1997) Science 275, Acad. Sci. U. S. A. 97, 9834 –9839 661– 665 26. Neville, C., Gonzales, D., Houghton, L., McGrew, M. J., and Rosenthal, N. 2. Franke, T. F., Kaplan, D. R., and Cantley, L. C. (1997) Cell 88, 435– 437 (1996) Dev. Genet. 19, 157–162 3. Songyang, Z., Baltimore, D., Cantley, L. C., Kaplan, D. R., and Franke, T. F. 27. Kerr, S. M., Taggart, M. H., Lee, M., and Cooke, H. J. (1996) Hum. Mol. Genet. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 11345–11350 5, 1139 –1148 4. Matsui, T., Li, L., del Monte, F., Fukui, Y., Franke, T. F., Hajjar, R. J., and 28. Duan, C., Liimatta, M. B., and Bottum, O. L. (1999) J. Biol. Chem. 274, Rosenzweig, A. (1999) Circulation 100, 2373–2379 37147–37153 5. Matsui, T., Tao, J., del Monte, F., Lee, K. H., Li, L., Picard, M., Force, T. L., 29. Jonassen, A. K., Sack, M. N., Mjos, O. D., and Yellon, D. M. (2001) Circ. Res. Franke, T. F., Hajjar, R. J., and Rosenzweig, A. (2001) Circulation 104, 89, 1191–1198 330 –335 30. Friddle, C. J., Koga, T., Rubin, E. M., and Bristow, J. (2000) Proc. Natl. Acad. 6. Cook, S. A., Sugden, P. H., and Clerk, A. (1999) Circ. Res. 85, 940 –949 Sci. U. S. A. 97, 6745– 6750 7. Proud, C. G., and Denton, R. M. (1997) Biochem. J. 328, 329 –341 31. Clemmons, D. R. (1998) Mol. Cell. Endocrinol. 140, 19 –24 8. Brunet, A., Datta, S. R., and Greenberg, M. E. (2001) Curr. Opin. Neurobiol. 32. Perks, C. M., McCaig, C., and Holly, J. M. (2000) J. Cell. Biochem. 80, 248 –258 11, 297–305 33. Roschier, M., Kuusisto, E., Suuronen, T., Korhonen, P., Kyrylenko, S., and 9. Kuhn, I., Bartholdi, M. F., Salamon, H., Feldman, R. I., Roth, R. A., and Salminen, A. (2001) J. Neurochem. 76, 11–20 Johnson, P. H. (2001) Physiol. Genomics 7, 105–114 34. McCaig, C., Perks, C. M., and Holly, J. M. (2002) J. Cell. Biochem. 84, 784 –794 10. Barthel, A., Okino, S. T., Liao, J., Nakatani, K., Li, J., Whitlock, J. P., Jr., and 35. Apstein, C. S., Gravino, F. N., and Haudenschild, C. C. (1983) Circ. Res. 52, Roth, R. A. (1999) J. Biol. Chem. 274, 20281–20286 515–526 11. Jiang, B.-H., Zheng, J. Z., Aoki, M., and Vogt, P. K. (2000) Proc. Natl. Acad. 36. Malhotra, R., and Brosius, F. C., III (1999) J. Biol. Chem. 274, 12567–12575 Sci. U. S. A. 97, 1749 –1753 37. Knight, J. (2001) Nature 410, 860 – 861 12. Pugazhenthi, S., Nesterova, A., Sable, C., Heidenreich, K. A., Boxer, L. M., 38. Bernal-Mizrachi, E., Wen, W., Stahlhut, S., Welling, C. M., and Permutt, M. A. Heasley, L. E., and Reusch, J. E. (2000) J. Biol. Chem. 275, 10761–10766 (2001) J. Clin. Investig. 108, 1631–1638 13. Biggs, W. H., III, Meisenhelder, J., Hunter, T., Cavenee, W. K., and Arden, 39. Bodine, S. C., Stitt, T. N., Gonzalez, M., Kline, W. O., Stover, G. L., Bauerlein, K. C. (1999) Proc. Natl. Acad. Sci. U. S. A. 96, 7421–7426 R., Zlotchenko, E., Scrimgeour, A., Lawrence, J. C., Glass, D. J., and 14. Brunet, A., Bonni, A., Zigmond, M. J., Lin, M. Z., Juo, P., Hu, L. S., Anderson, Yancopoulos, G. D. (2001) Nat. Cell Biol. 3, 1014 –1019 M. J., Arden, K. C., Blenis, J., and Greenberg, M. E. (1999) Cell 96, 857– 868 40. Godecke, A., and Schrader, J. (2000) Basic Res. Cardiol. 95, 492– 498 15. Tang, E. D., Nunez, G., Barr, F. G., and Guan, K. L. (1999) J. Biol. Chem. 274, 41. Fishman, G. I. (1998) Circ. Res. 82, 837– 844 16741–16746 42. McPherron, A. C., and Lee, S. J. (1997) Proc. Natl. Acad. Sci. U. S. A. 94, 16. Kitaura, J., Asai, K., Maeda-Yamamoto, M., Kawakami, Y., Kikkawa, U., and 12457–12461 Kawakami, T. (2000) J. Exp. Med. 192, 729 –740 17. Du, K., and Montminy, M. (1998) J. Biol. Chem. 273, 32377–32379 43. McPherron, A. C., Lawler, A. M., and Lee, S. J. (1997) Nature 387, 83–90 44. Sharma, M., Kambadur, R., Matthews, K. G., Somers, W. G., Devlin, G. P., 18. Ozes, O. N., Mayo, L. D., Gustin, J. A., Pfeffer, S. R., Pfeffer, L. M., and Donner, D. B. (1999) Nature 401, 82– 85 Conaglen, J. V., Fowke, P. J., and Bass, J. J. (1999) J. Cell. Physiol. 19. Romashkova, J. A., and Makarov, S. S. (1999) Nature 401, 86 –90 180, 1–9

Journal

Journal of Biological Chemistry – Unpaywall

Published: Jun 1, 2002

There are no references for this article.