Developing a Strategy to Define the Effects of Insulin-Like Growth Factor-1 on Gene Expression Profile in Cardiomyocytes
Abstract—Insulin-like growth factor (IGF)-1 activates intracellular signaling pathways and regulates myocardial structure and function. This study used DNA microarray to define the effects of IGF-1 on gene expression in cardiomyocytes. Despite DNA microarray becoming a popular tool for profiling gene expression, the specificity of DNA microarray results is rarely addressed. Our data showed that the specificity of a DNA microarray study can be increased by repetitive experiments and by excluding minimally expressed genes. In this study, the false-positive rates were reduced to <0.2%. Future DNA microarray studies should incorporate a proper strategy to minimize false-positive results. IGF-1 modulates the expression of genes in 17 functional categories, but most genes clustered around the regulation of intracellular signaling, cell cycle, transcription/translation, cellular respiration and mitochondrial function, cell survival, ion channels and calcium signaling, and humoral factors. To further explore whether extracellular signal–regulated kinase (ERK) and phosphatidylinositol (PI) 3 kinase specifically regulate different sets of genes, the effects of IGF-1 were inhibited with PD98059 or LY294002. The results showed that the majority of genes regulated by IGF-1 required activation of both ERK and PI 3 kinase. Thus, PI 3 kinase and ERK coordinately mediate the transcriptional regulatory effects of IGF-1 in cardiac muscle cells. These findings provide novel insight into how IGF-1 signaling modulates the programming of cardiac muscle gene expression.
- insulin-like growth factor
- cardiac muscle
- gene expression
- phosphatidylinositol 3 kinase
- mitogen-activated protein kinase
Insulin-like growth factor (IGF)-1 plays important roles in cardiac muscle biology.1 2 The actions of IGF-1 are mediated through activation of intracellular signaling pathways.2 The two major pathways of IGF-1 receptor signaling are the phosphatidylinositol (PI) 3 kinase pathway and the mitogen-activated protein (MAP) kinase pathway.3 Activation of intracellular signaling pathways may ultimately lead to modulation of gene expression and regulation of cell function. Although a number of genes are known to be upregulated or downregulated by IGF-1, such as c-jun and myosin heavy chain,3 4 these genes likely represent a small fraction of the genes that are regulated by IGF-1. Because the biological actions of IGF-1 are mediated in part through modulation of gene expression, it will be important to systemically define the effects of IGF-1 on gene expression.
The DNA microarray is a powerful technology that offers comprehensive profiling of gene expression.5 Like any new technology, the microarray is not free from experimental errors, and it may inherit the unwanted variables introduced by the samples applied to the microarray.6 Existing literature involving DNA microarray study rarely address the specificity of microarray data. A lack of information on the specificity of microarray data may impede the development of adequate strategies for data analysis and, thus, poses a serious problem in the experiments involving DNA microarray.
In the present study, we used primary cardiomyocytes as a model to investigate whether the DNA microarray spotted on nylon membrane can be reliably used to profile the effects of IGF-1 on cardiac muscle gene expression. The results showed that multiple experiments and appropriate analysis strategies are needed to avoid false-positive results. Once an appropriate analysis strategy was established, our results showed that IGF-1 modulated the expression of many genes involved in the regulation of diverse biological functions. Moreover, the regulatory effects of IGF-1 on the majority of these genes require concomitant activation of phosphatidylinositol (PI) 3 kinase and extracellular signal–regulated kinase (ERK) signaling pathways.
Materials and Methods
Sprague-Dawley rats were obtained from Simonsen Laboratory (Gilroy, Calif). IGF-1 was a gift from Genentech (South San Francisco, Calif). PD098059 and LY294002 were purchased from Biomol. DNase I and Superscript II RT were obtained from GIBCO-BRL. Salmon testis DNA was purchased from Pharmacia. [α-32P]ATP was from Amersham. Other chemicals were purchased from Sigma Chemical Co or Fisher. cDNA Rat 1.2 Microarray, Synthesis Primer Mix, ExpressHyb Hybridization Solution, and AtlasImage 1.01a analysis software were from Clontech.
Primary Cardiomyocyte Culture
Primary cultures of rat cardiomyocytes were prepared from the cardiac ventricles of Sprague-Dawley neonates as previously described.7 All surgical procedures were approved by the institutional review board at the University of California, Irvine. At the time of experiments, these preparations contained >95% cardiomyocytes. Myocytes were cultured in 100-mm dishes in DMEM containing 10% FBS. To study the effects of IGF-1 on gene expression, primary cardiomyocytes were rinsed twice with serum-free DMEM, serum-deprived overnight, and then incubated with IGF-1 (10−6 mmol/L) or vehicles for indicated time intervals. A specific PI 3 kinase inhibitor (LY294002) and a specific MAP kinase kinase (MEK) inhibitor (PD098059) were added to culture medium 45 minutes before the addition of IGF-1 to inhibit PI 3 kinase or MEK signaling when indicated.
Extraction and Processing of RNA
The attached cells were scraped, and the detached cells floating in the medium were collected with centrifugation (4000g, 4°C, 10 minutes). The cells were washed once and lysed; total RNA was isolated with the Rneasy Mini Kit according to the manufacturer’s instructions (Qiagen). RNA samples were treated with DNase I (1 U/μL) to remove any residual DNA, and the quality of RNA was confirmed with denaturing formaldehyde/agarose gel electrophoresis. Each RNA sample was pooled from the RNAs harvested from three to four 100-mm dishes of primary cardiomyocytes.
Labeling of cDNA Probes and Hybridization to Microarray
Gene-specific primers were used to generate labeled cDNA probes. In brief, total RNA (5 μg) was mixed with gene-specific primer mixture and heated to 70°C for 10 minutes and reverse-transcribed into radiolabeled cDNA probes with SuperScript II in the presence of [α-32P]dATP, dNTPs, and dithiothreitol for 50 minutes at 42°C. Reverse transcription was terminated by heating to 70°C for 15 minutes. Labeled cDNAs were purified with QIAquick Nucleotide Removal Kit (Qiagen) to remove unincorporated [32P]dATP and small cDNA fragments. This protocol typically yields >2×106 cpm of labeled cDNA probes from each sample. The Rat 1.2 cDNA Microarrays were first soaked in 0.5% SDS at 85°C for 2 minutes and rinsed with distilled deionized water. The microarrays were then prehybridized in 1× ExpressHyb Hybridization Solution in the presence of 20 μg/mL heat-denatured salmon testis DNA at 68°C for 4 hours. 32P-labeled cDNA probes were then added to the solution and hybridized to the array at 68°C for 16 hours. Then, the microarrays were washed four times with 1× SSC+1% SDS (68°C, 30 minutes each), one time with 0.1×SSC+0.5% SDS at 68°C for 30 minutes, and finally, one time with 2× SSC at room temperature for 5 minutes. The arrays were exposed to the Storage Phosphor Screen from Molecular Dynamics.
Microarray Data Analysis and Statistics
Microarray signals on the Storage Phosphor Screen were read with ImageQuant 4.0 Software and PhosphorImager (Molecular Dynamics). The scanned images were aligned and analyzed with the AtlasImage 1.01a software. Because background signals were generally homogeneous in these arrays, a globally averaged background from each microarray membrane was subtracted from the density in each specific array signal spot. If the signal intensity in specific array spot is equal or less than the background intensity, the signal intensity in this spot is defined as zero. All array spots that contain zero readings in the control and IGF-I–treated samples were excluded from final analysis, because the expression of these genes was either silent or undetectable in cardiomyocytes. We have visually inspected the images in all array spots in every experiment to ensure that the signal density is not the result of artifact or high background signal. The relative level of expression for each gene was calculated as a fraction of the total signal intensity on the entire membrane. The ratio of expression levels between the control and IGF-I–treated samples was obtained from each set of the experiment. A differentially expressed gene is defined by a ratio of IGF-1 treatment/control >1.5 or <0.67. For statistical analysis, the differences between the control and IGF-1–treated samples were determined with a 2-tailed t test via the Internet (Cyber T), and a value of P<0.05 was considered statistically significant.
Specificity of Data Obtained From Microarray
Because of the high costs of microarray study, most investigators elected to limit the number of repetitive array experiments. It is possible that a limited number of repetitions may lead to false-positive results. To determine whether microarray study is associated with experimental artifacts and whether the artifacts can be offset by adequate analytical strategies, we first compared the results of microarray study derived from independent RNA samples extracted from control cardiomyocytes. Because these control cardiomyocytes were grown and harvested under the same protocol, any differential expression of a given gene in two different sets of control RNA can be interpreted as false-positive results. We used the following criteria to determine whether the expression of a gene is upregulated or downregulated: (1) the difference between two experimental settings was statistically significant by t test, or (2) differential expression was consistently upregulated or downregulated in five of six experiments. The second criterion was added because the t test usually underestimates the significance of difference when the sample size is small. Insufficient repetition of a given experiment leads to low sensitivity, and with the high cost of microarray, it is unlikely that a microarray experiment can be repeated many times to reach high sensitivity. In a typical microarray study with a small sample size, relying on the t test alone will lead to an underestimation of the number of genes that are truly differentially expressed. Using the above criteria, we compared the gene expression profile in two paired sets of control RNA samples, six samples in each set. The Figure⇓ shows that there is a linear relationship of gene expression between these two sets of control RNAs. Two differentially expressed genes between these two sets of control samples were found (Figure⇓), indicating that with the use of the above approach, the false-positive rate is very low (<0.2%).
Further analysis revealed that two factors, the number of repetitive experiments and the level of gene expression, might increase the risk of obtaining false-positive data. Table 1⇓ shows that the number of differentially expressed genes between two sets of controls (false positive) was in proportion to the number of RNA samples included in the microarray study. These data suggest that in each experimental group, at least six independent RNA samples are needed to minimize the risk of a false-positive result. The levels of expression also contributed to the specificity of microarray results. Although the correlation has been linear, as shown in the Figure⇑, a higher level of variation was seen in those genes that were minimally expressed. When minimally expressed genes were included in the analysis, the risk of false-positive results was increased (Table 2⇓). The background signal intensity on these microarray membranes is ≈1.0×10−4 of the total signal on membrane; therefore, the above data indicate that reliable results are more likely to be obtained when the expression level is >4 times over background.
Regulation of Gene Expression by IGF-1
To define the effects of IGF-1 on gene expression, cardiomyocytes were stimulated with IGF-1 at various time intervals as described in Materials and Methods. No visible morphological changes were observed in cardiomyocytes after 6 hours of IGF-1 treatment. Radiolabeled cDNA probes were generated from the RNA extracted from control and IGF-1–stimulated cardiomyocytes and hybridized to microarray membranes. Using the analysis strategy described above with six independent sets of RNA in each experimental group and a background cutoff at 4×10−4, we have identified 68 genes (5.7%) that were differentially expressed on IGF-1 stimulation (Table 3⇓). The time course of gene expression showed that IGF-1 regulated the coordinated expression of cardiac muscle genes. Most differentially expressed genes were found after 2 hours of IGF-1 incubation. These genes are involved in diverse cellular and biochemical functions. IGF-1–regulatable genes can be categorized into 17 major groups: cell cycle regulators, transcription factors, RNA synthesis/processing, protein processing, proto-oncogenes, tumor suppressors, signal transduction, cellular respiration and energy production, stress response and antioxidation, ion channels, cell surface receptors, cell survival, cytoskeleton structure, transporter, humoral factors, carbohydrate metabolism, and cell-cell interaction. However, the majority of these genes are involved in the regulation of intracellular signaling, cell cycle, transcription/translation, cellular respiration and mitochondrial function, cell survival, ion channels and calcium signaling, and humoral factors.
Most genes were upregulated; only six genes were downregulated by IGF-1. Some genes were persistently downregulated or upregulated, whereas some other genes were only upregulated or downregulated at specific time points. Many genes that are known to modulate myocardial structure and function were modulated by IGF-1, such as several proteins related to calcium signaling, ion channels, heat shock proteins, G proteins, copper-zinc superoxide dismutase-1, plasminogen activator inhibitor-1, metalloproteinase inhibitor-3, vascular endothelial growth factor (VEGF), natriuretic peptide, c-jun, inhibitor of differentiation-1, and a list of genes related to mitochondrial metabolism and energy production. Several genes related to cell cycle regulation were coordinately regulated. For example, cyclin D1, cyclin D2, cyclin D3, proliferating cell nuclear antigen, v-fos transformation effector (Fte-1), and casein kinase II were upregulated at various time intervals on IGF-1 stimulation. Other signal transduction proteins and proto-oncogenes that are regulated by IGF-1 in cardiomyocytes have also been previously implicated in the regulation of cell cycle regulation, suggesting that regulation of the cell cycle is a major function of IGF-1.
IGF-1 Regulation of Gene Expression Involves Coordinated Activation of PI 3 Kinase and MAP Kinase Pathways in Cardiac Muscle
To determine whether IGF-1 regulation of gene expression involves activation of PI 3 kinase or ERK signaling pathways, we have used chemical inhibitors of PI 3 kinase (LY294002) and MEK (PD98059) to dissect the roles of PI 3 kinase and ERK pathways in IGF-1 regulation of gene expression in cardiac muscle cells (Table 4⇓). Because two thirds of the genes modulated by IGF-1 occurred after 2 hours of stimulation, these experiments were carried out with 2 hours of IGF-1 incubation. To our surprise, the effect of IGF-1 on 31 genes can be inhibited either by LY294002 alone or by PD98059 alone, indicating that IGF-1 actions on the expression of these genes require activation of both PI 3 kinase and ERK pathways. Nevertheless, the effects of IGF-1 on nine genes were inhibited by LY294002 and not by PD98059, suggesting that the effects of IGF-1 on these genes are dependent on PI 3 kinase alone. Conversely, IGF-1 regulation of transducin-β2 was blocked by PD98059 but not by LY294002, suggesting that IGF-1 regulation of transducin-β2 expression was dependent on activation of the MEK/ERK signaling pathway alone. The effects of IGF-1 on three genes cannot be blocked by either LY294002 or PD98059, suggesting that additional IGF-1 signaling pathways independent of PI 3 kinase and ERK were involved in the regulation of these genes.
These data suggest that PI 3 kinase and MEK/ERK are major signaling pathways mediating the IGF-1 regulation of gene expression in cardiac muscle cells. However, the IGF-1 regulation of cardiac gene expression may require coordinated activation of more than one signaling pathway because IGF-1 regulation of many genes requires the activation of both pathways.
Pitfalls in DNA Microarray Experiment
DNA microarrays provide a powerful tool for systemic analysis of gene expression in response to growth factor actions.8 9 However, the specificity of DNA microarray data was seldom explored in the literature, and the risk of false-positive results was hardly ever discussed. Our results clearly demonstrate the potential risk of obtaining false-positive results if adequate analytical strategy is not implemented. The number of repetitive experiments required and the cutoff point for minimally expressed genes should be carefully examined at the beginning of a microarray project.
Manufacturers of DNA microarrays usually recommend 2- or 3-fold changes as “significant changes” of gene expression.8 9 10 11 However, our data do not support such a recommendation. From our analysis, a false-positive gene can be differentially expressed in the range of 2-fold or 3-fold. Conversely, a 1.5-fold change in gene expression after IGF-1 treatment can be highly consistent and statistically significant. These data indicate that using 2-fold or 3-fold changes as lone criteria to judge whether gene expression is altered in a microarray study is not reliable and may underestimate the number of genes differentially expressed. When the same RNA sample was hybridized to two different microarrays, ≈95% of the gene expression pattern can be reproduced. Thus, the reproducibility appears quite good. However, we were not able to define the exact sensitivity (the chance of positive being a true positive) of our microarray data, because the regulatory effects of IGF-1 on the majority of genes included in this array were not previously known.
The vast majority of published microarray studies did not report an analysis of specificity; a lack of such information may dampen the validity of microarray data and mislead future research direction. One solution is to perform detailed specificity analysis at the beginning of a microarray project and to report the results of such an analysis. Because more and more investigators will be using this revolutionary tool to study gene expression, there is an urgent need to explore the feasibility of a basic format to report microarray data. An ideal format should include at least the following key elements: the strategies used to exploit data specificity, statistical justification for the chosen strategy, and the risk of false-positive results under the experimental setting in each study.
IGF-1 Modulation of Gene Expression in Cardiomyocytes
IGF-1 modulates multiple aspects of cardiovascular function, and the data presented in the present study clearly reflect diverse biological actions of IGF-1 in cardiac muscle cells. The majority of the genes modulated by IGF-1 reported in the present study had not been shown in the past; thus, novel actions of IGF-1 on gene expression are represented. We have searched the literature and found that among the 68 genes that were regulated by IGF-1 in this study, 15 genes were modulated by IGF-1 in previous studies, mostly in different experimental systems. However, one of the 68 genes (bone morphogenetic protein-4) had a different pattern of regulation on IGF-1 stimulation (upregulation versus downregulation) in human dental pulp fibroblasts, a cell type vastly different from cardiomyocytes.
Several signaling proteins were induced by IGF-1 in cardiac muscle; interestingly, the protein products of these genes may function downstream from IGF-1 receptor signaling. A-Raf, an upstream serine/threonine kinase of ERK pathways,12 plays a pivotal role in mediating growth-promoting effects. Casein kinase II is a highly conserved ubiquitous serine/threonine kinase that participates in the regulation of cell cycle, cell homeostasis, and cell survival.13 Signal transducer(s) and activator(s) of transcription (STAT)3 activates gene transcription and plays a role during cell differentiation and survival.14 In addition, 14-3-3 protein has been implicated in the regulation of the Raf/ERK signaling pathway.14 These findings suggest that IGF-1 may have dual effects on these signaling pathways. An acute action that activates these intracellular signaling proteins through phosphorylation cascades, and a delayed effect that modulates their expression via acute activation of signaling.
The G1 cyclins, including D-type cyclins, are positive regulators of cell cycle progression from G1 to S phase.15 In addition to cyclin Ds, our data showed that IGF-1 upregulated proliferating cell nuclear antigen (a protein involved in DNA synthesis), c-jun (a component of activator protein-1), HNRNP-K (a transcription factor for c-Myc), STAT3 (a key element in the Janus kinase/STAT pathway), and ID-1 (a helix-loop-helix transcription factor). The transcriptional regulatory effects of IGF-1 on these molecules may partially explain the well-known actions of IGF-1 on DNA synthesis and mitogenesis. Although whether cardiomyocyte DNA synthesis and proliferation occur in the adult heart remains a controversial issue, there is good evidence suggesting that the development of cardiac hypertrophy involves perturbation of cell cycle controls16 and that chronic infusion of IGF-1 leads to mild cardiac hypertrophy.17 It is possible that IGF-1 regulation of cell cycle control may contribute to the pathophysiological actions of IGF-1 in the heart.
Series of genes involved in the regulation of cellular respiration and energy production are modulated by IGF-1. Oxidative metabolism is the fundamental mechanism through which energy, in the form of ATP, is provided to the cardiac muscle cells.18 Genetic defects of mitochondrial oxidative phosphorylation and fatty acid oxidation may lead to the development of cardiomyopathy,19 and mitochondrial dysfunction had been proposed to contribute to the decline of cardiac function in elderly patients.20 Moreover, abnormal mitochondrial structure and function have been observed in experimental and human cardiomyopathy, and decreased mitochondrial respiration has been observed in an animal model of heart failure.21 It is possible that the effects of IGF-1 on mitochondrial enzyme expression may lead to the modulation of mitochondrial function in the heart.
Detoxifying proteins are responsible for metabolizing toxins and drugs into less adverse metabolites. Similarly, antioxidant enzymes, which catalyze peroxides into nontoxic molecules, prevent cells from excessive oxidative stress. The fact that IGF-1 stimulates the gene expression of detoxifying proteins and antioxidant enzymes reflects the protective role of IGF-1 on cardiomyocyte survival. IGF-1 has been shown to regulate the Bcl-2 family of proteins,22 and microarray results indicate that IGF-1 increased the expression of the antiapoptotic protein Bcl-2. IGF-1 also increased the expression of cyclin D and STAT3, two proteins that have been shown to increase cell resistance to the induction of apoptosis.23 24 These results are consistent with numerous previous studies indicating that IGF-1 increases cardiac muscle survival in experimental models of heart failure.25 26
Previous studies have demonstrated the inotropic effects of IGF-1 in normal animals and experimental heart failure.2 27 Calcium and other ion channels play important roles in the regulation of myocardial contractility. In the present study, we found that IGF-1 regulates the expression of the L-type cardiac calcium channel, sodium channel, and Na+-H+ exchange protein. Moreover, calmodulin was also upregulated by IGF-1. These data raised the possibility that IGF-1 may exert positive inotropic effects through modulation of sodium channels and provide further evidence that that IGF-1 modulation of calcium signaling could have contributed to its inotropic effects.2 27
Several growth factors and humoral factors were modulated by IGF-1, some of which are known to modulate myocardial function. IGF-1 is known to induce VEGF expression in various cells.28 29 In the present study, we also found that the expression of glioma-derived vascular endothelial cell growth factor, a member of the VEGF family, could be stimulated by IGF-1. Some other humoral factors regulated by IGF-1, such as SMAD5 and plasminogen activator inhibitor-1, have also been implicated in angiogenesis.30 31 These findings suggest that IGF-1 may promote angiogenesis in cardiac muscle. PAIs belong to a family of proteins that exert stimulatory effects on cell proliferation during developmental stage. IGF-1 has been previously reported to increase PAI-1 mRNA levels in Hep G2 cells,32 mainly through prolonging its half-life at the post-transcriptional level.
Independent and Coordinated Regulation of Gene Expression by Intracellular Signaling Pathways
IGF-1 and other peptide growth factors bind to specific cell-surface receptors and trigger multiple intracellular signaling pathways. A great deal of research during the last decade had been focused on the specificity of each signaling pathway in relation to their unique biological actions. However, recent studies indicate that multiple signaling pathways can be coordinately activated to regulate a biological action. For example, insulin activation of glucose transporter-4 translocation and of glucose transport requires activation of the PI 3 kinase pathway and a PI 3 kinase–independent pathway.33 There is also evidence indicating that activation of the PI 3 kinase and ERK pathways synergistically regulates insulin receptor trafficking and cell survival.34 35 It is not yet clear how different signaling pathways, such as the PI 3 kinase pathway and ERK pathway, coordinately regulate cardiac muscle function. The present study provides a good argument that a significant fraction of IGF-1–regulatable genes are coordinately modulated through the activation of PI 3 kinase and MEK/ERK pathways. Nevertheless, the specificity of signaling pathway on gene expression indeed exists in cardiomyocytes because activation of PI 3 kinase or ERK by IGF-1 independently leads to specific modulation of mRNA levels in some genes. Thus, IGF-1 activation of PI 3 kinase and ERK signaling independently and coordinately mediates the IGF-1 regulation of gene expression.
This study was supported by grants from the National Heart, Lung, and Blood Institute, American Heart Association, and American Diabetes Association (to Dr Wang). The authors wish to thank Dr Wesley Hatfield for helpful data discussion.
Original received November 1, 2000; revision received April 24, 2001; accepted April 24, 2001.
- © 2001 American Heart Association, Inc.
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