Molecular Pharmacology Fast Forward. Published on July 22, 2004 as DOI: 10.1124/mol.104.000950 Molecular PharmacologyThis article has Fastnot been Forward. copyedited Publishedand formatted. Theon finalJuly version 22, 2004 may differ as doi:10.1124/mol.104.000950from this version. MOLPHARM/2004/000950

Title

Full title: Transcriptional Response to Corticotropin-releasing Factor in AtT-20 Cells.

Authors: Pieter J. Peeters, Hinrich W. Göhlmann, Ilse Van den Wyngaert, Sigrid M.

Swagemakers, Luc Bijnens, Stefan U. Kass and Thomas Steckler.

Downloaded from Primary laboratory of origin: Johnson & Johnson Pharmaceutical Research and Development,

a division of Janssen Pharmaceutica N.V., Turnhoutseweg 30, 2340 Beerse, Belgium molpharm.aspetjournals.org at ASPET Journals on September 25, 2021

1

Copyright 2004 by the American Society for Pharmacology and Experimental Therapeutics. Molecular Pharmacology Fast Forward. Published on July 22, 2004 as DOI: 10.1124/mol.104.000950 This article has not been copyedited and formatted. The final version may differ from this version.

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Running title

a) Running title: Transcriptional response to CRF b) Corresponding author: Pieter Peeters, Johnson & Johnson Pharmaceutical Research and

Development, a division of Janssen Pharmaceutica N.V., Turnhoutseweg 30, 2340 Beerse,

Belgium. Tel. +32(0)14606648, Fax. +32(0)14605737, e-mail [email protected] c) manuscript includes Downloaded from 23 text pages,

2 tables,

7 figures, molpharm.aspetjournals.org

47 references,

250 words in abstract,

737 words in introduction,

1213 words in discussion. at ASPET Journals on September 25, 2021 d) non-standard abbreviations: ACTH, adrenocorticotropin; CAMKII, Ca2+/calmodulin- dependent kinase II; CCK, cholecystokinin; CFA, correspondence factor analysis; CREB,

Ca2+/cAMP response element-binding protein; CREM, cAMP responsive element modulator;

CRF, corticotropin-releasing factor; CRFR1, corticotropin-releasing factor receptor 1;

CRFR2, corticotropin-releasing factor receptor 2; CT, calcitonin; Fgfr2, fibroblast growth factor receptor 2; Hey1, hairy/enhancer-of-split related 1; Mif, macrophage migration- inhibitory factor gene; Mig6, mitogen-inducible gene 6; Nfil3, nuclear factor regulated by interleukin 3; PCA, principal component analysis; Pde4b, cAMP specific phosphodiesterase

4B; Pi3k, regulatory subunit of phosphatidylinositol 3-kinase, p85; PKA, protein kinase A;

Pse, prostate specific ets transcription factor; Ramp3, receptor (calcitonin) activity modifying protein 3; RGS2, regulator of G-protein signaling 2; Sgk, serum/glucocorticoid regulated kinase; SPM, spectral map analysis.

2 Molecular Pharmacology Fast Forward. Published on July 22, 2004 as DOI: 10.1124/mol.104.000950 This article has not been copyedited and formatted. The final version may differ from this version.

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Abstract

Corticotropin-releasing factor (CRF) plays a central role in regulation of the hypothalamic- pituitary-adrenal axis, mediating endocrine and behavioral responses to various stressors. Two high affinity receptors for CRF have been described. Although many of the intracellular signaling pathways activated by CRF have been extensively studied, our knowledge of transcriptional responses downstream of the CRF receptor 1 (CRFR1) is still limited. To elucidate gene networks regulated by CRF and CRFR1, we applied microarray technology to Downloaded from explore transcriptional response to CRF stimulation. Therefore mouse pituitary derived AtT-

20 cells were continuously exposed to CRF either in the presence or absence of a specific

CRFR1 antagonist R121919. Transcriptional responses to different treatments were studied in molpharm.aspetjournals.org a time course ranging from 0.5 to 24 hours. Microarray data were analyzed using classical microarray data analysis tools such as correspondence factor analysis, cluster analysis and fold change filtering. Furthermore spectral map analysis was applied, a recently introduced unsupervised multivariate analysis method. A broad and transient transcriptional response to at ASPET Journals on September 25, 2021 CRF was identified that could be blocked by the antagonist. This way several known CRF- induced target genes and novel CRF responsive genes were identified. These include transcription factors such as cAMP responsive element modulator (CREM, 7X increased), secreted peptides such as cholecystokinin (1.5X) and proteins involved in modulating intracellular signaling, for example regulator of G-protein signaling 2 (Rgs2, 11X).

Upregulation of many of these genes can be explained as negative feedback, attenuating CRF activated pathways. Additionally, spectral map analysis proved to be a promising new tool for microarray data analysis.

3 Molecular Pharmacology Fast Forward. Published on July 22, 2004 as DOI: 10.1124/mol.104.000950 This article has not been copyedited and formatted. The final version may differ from this version.

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Introduction

Corticotrophin releasing factor (CRF), a 41-amino acid polypeptide plays a central role in the regulation of the hypothalamic-pituitary-adrenal axis, mediating neuroendocrine, autonomic and behavioural responses to various stressors. Hypothalamic neurons from the parvocellular part of the paraventricular nucleus release CRF into the hypophyseal portal system in response to stress, stimulating the secretion and biosynthesis of adrenocorticotropin (ACTH) from pituitary corticotrophs, subsequently leading to increased adrenal glucocorticoid release Downloaded from (Steckler, 2001). Two high affinity receptors for CRF have been described, CRFR1 and

CRFR2, both of which exist in several splice variant forms (Dautzenberg et al., 2001).

CRFR1 expression is abundant in the brain and pituitary and is limited in the periphery molpharm.aspetjournals.org

(Steckler and Holsboer, 1999). In contrast to CRFR1, the expression pattern of CRFR2 is discrete in the brain and more widespread in the periphery. The intracellular signaling pathways activated by CRF in the anterior pituitary have been extensively studied (Bilezikjian and Vale, 1983;Aguilera et al., 1988;Rossant et al., 1999). Activation of the receptor by CRF at ASPET Journals on September 25, 2021 results in Gs-mediated stimulation of adenyl cyclase leading to increased levels of intracellular cAMP (Stalla et al., 1988). These high levels of cAMP lead to the activation of the cAMP dependent protein kinase A (PKA) (Litvin et al., 1984). In addition to augmenting production of cAMP, it has been shown that CRF triggers a persistent membrane depolarization via PKA-dependent closure of K+ channels (Lee and Tse, 1997). This membrane depolarization in turn activates Ca2+ entry through L-type and P-type voltage-gated

Ca2+ channels and causes a continuous cytosolic Ca2+ concentration increase. As a result of this, exocytosis via secretion vesicles located near the plasma membrane is stimulated (Tse and Lee, 2000).

Increased cytosolic levels of cAMP and Ca2+ lead to the activation of several other downstream effectors of CRF receptors. In addition to the above mentioned PKA, additional kinases such as Ca2+/calmodulin-dependent kinase II (CAMKII) and p42/p44 mitogen activated kinases are activated upon stimulation with CRF. As a result Ca2+/cAMP response

4 Molecular Pharmacology Fast Forward. Published on July 22, 2004 as DOI: 10.1124/mol.104.000950 This article has not been copyedited and formatted. The final version may differ from this version.

MOLPHARM/2004/000950 element-binding protein (CREB) transcription factor is phosphorylated, in turn regulating the transcription of genes containing the Ca2+/cAMP response element (CRE) (Rossant et al.,

1999). Examples of such genes include c-fos (Boutillier et al., 1991), macrophage migration- inhibitory factor gene Mif (Waeber et al., 1998), orphan nuclear receptors Nur77 and Nurr1

(Murphy and Conneely, 1997). For the latter it has been recently shown that CRF signalling leads to the induction of mRNA through PKA and CAMKII dependent mechanisms whereas

Nurr77 transactivation through phosphorylation is dependent on MAPK regulation Downloaded from (Kovalovsky et al., 2002).

In addition to intracellular protein kinases, Ca2+ and cAMP, CRH receptors modulate a plethora of other important signaling molecules such as nitric oxide synthase (NOS), guanylyl molpharm.aspetjournals.org cyclase, Fas and Fas ligand, procaspase-3 pathways and suppress activities of glycogen synthase kinase 3 and nuclear factor-kappaB (Dermitzaki et al., 2002;Cantarella et al.,

2001;Kostic et al., 2001;Radulovic et al., 2003). It is not always clear whether these effects are mediated via the CRHR1 or CRHR2 subtype. In case of nitric oxide synthase it seems that at ASPET Journals on September 25, 2021 the divergent effects of CRH on different cell lines can be explained by different expression of either subtype of its receptor (Cantarella et al., 2001). A long the same line, accumulating evidence suggests tissue-specific coupling to G proteins and activation of different signaling cascades. In some tissues CRFR1 may couple to Gq and activate the phospholipase C-protein kinase C cascade (Grammatopoulos et al., 2000).

AtT-20 cells have been studied intensively as a cellular model of corticotrophs. As such, many of the important players of the signaling cascade downstream of CRFR1 have been identified and studied in this cell line. Although several aspects of CRFR1 signaling have been extensively studied, at a transcriptional level our insight is limited. The aim of this study was to explore the transcriptional response to continuous CRF exposure in the mouse pituitary corticotroph-derived adenoma cell line AtT-20 at the genome level using oligonucleotide microarrays and quantitative PCR. We report on novel CRF-responsive genes that are transiently induced after CRF exposure, an induction that can be suppressed by a CRFR1 specific antagonist R121919. Many of the induced transcripts encode proteins that would

5 Molecular Pharmacology Fast Forward. Published on July 22, 2004 as DOI: 10.1124/mol.104.000950 This article has not been copyedited and formatted. The final version may differ from this version.

MOLPHARM/2004/000950 exert a negative feedback on the CRFR1 signaling possibly contributing to the transient nature of the induction. The use of spectral map analysis of microarray data revealed the presence of both time and CRF related effects in transcriptional responses to prolonged CRF exposure. Our results demonstrate the power of this projection method in microarray data analysis. Downloaded from molpharm.aspetjournals.org at ASPET Journals on September 25, 2021

6 Molecular Pharmacology Fast Forward. Published on July 22, 2004 as DOI: 10.1124/mol.104.000950 This article has not been copyedited and formatted. The final version may differ from this version.

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Material and Methods

Cell culture and sample preparation. AtT-20 cells were purchased from ATCC and were maintained at 37°C in 5% CO2 in humidified air in Dulbecco’s modified Eagle’s medium

(Invitrogen, Groningen, The Netherlands) containing 10 % fetal bovine serum, 5% horse serum and 4.5 g/L D-glucose. For experiments cells were seeded in 25 cm2 flasks. The medium was replaced 48 h later and cells were treated with either 0.1% DMSO, 1µM ovine

CRF (Sigma, St. Louis, MO) in DMSO, 1µM R121919 (3-[6-(dimethylamino)-4-methyl- Downloaded from pyrid-3-yl]-2,5-dimethyl-N,N-dipropyl-pyrazolo[2,3-a]pyrimidin-7-amine, Neurocrine

Biosciences, San Diego, CA) in DMSO or 1µM CRF +1µM R121919 in DMSO for 0, 0.5, 1,

2, 4, 8 and 24h in fresh medium. The dose of 1µM CRF was chosen because it was shown to molpharm.aspetjournals.org induce maximal response in an transactivation assay in AtT20 cells transfected with a luciferase reporter construct (data no shown). The incubation was stopped by aspirating the incubation medium and adding 3 ml Trizol (Invitrogen) for lysis of the cells. Total RNA was extracted using Trizol according to the instructions of the manufacturer. 100µg total RNA at ASPET Journals on September 25, 2021 was further purified using Qiagen Rneasy kit (Westburg, Leusden, The Netherlands) with

DNAseI treatment on column.

Microarray hybridisation. cRNA was prepared as follows. Reverse transcription was performed on 10µg of total RNA for 1h at 42°C using a T7-oligo(dT)24-primer and

SuperscriptII RT (Invitrogen). Second strand cDNA synthesis was done for 2 h at 16°C using

Escherichia coli DNA Polymerase I, DNA ligase and RNAseH (Invitrogen). After phenol- chloroform extraction using phase-lock gel (Eppendorf, Hamburg, Germany) in vitro transcription was performed for 6 h at 37°C using the Bioarray high-yield RNA transcript labelling kit with Biotin labelled ribonucleotides (Enzo Diagnostics, Farmingdale, NY). cRNA samples were purified on Qiagen Rneasy columns followed by fragmentation for 35 min at 95°C. cRNA yields were between 50 and 100µg. Samples were processed on

GeneChips (Affymetrix, Santa Clara, CA). In order to check the quality of each sample, 5µg of labelled cRNA was run on Test2-arrays. Actual experiments were performed on Murine

7 Molecular Pharmacology Fast Forward. Published on July 22, 2004 as DOI: 10.1124/mol.104.000950 This article has not been copyedited and formatted. The final version may differ from this version.

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Genome U74Av2a arrays, containing probe sets interrogating approximately 12,000 full- length mouse genes and EST clusters from the UniGene database (Build 74). Hybridisation was performed using 15µg cRNA for 16 h at 45°C under continuous rotation. Arrays were stained in Affymetrix Fluidics stations using Streptavidin/Phycoerythrin (SAPE) followed by staining with anti-streptavidin antibody and a second SAPE staining. Subsequently arrays were scanned with a Agilent Laserscanner (Affymetrix) and data were analysed with the

Microarray Suite Software 5.0 (Affymetrix). No scaling or normalization was performed at Downloaded from this stage. Quality of the experiment was assessed based on the percentages present calls across all samples which was on average 47.06±2.45%. The cytoplasmic ß-actin and GAPDH

5’/3’ ratios were 1.10±0.08 and 0.93±0.05 respectively. molpharm.aspetjournals.org

Data analysis and selection of genes.

Normalization. Genes which were called absent in all samples according to Affymetrix'

MAS 5.0 software (p-value of >0.06) were removed from further analysis. Raw intensities from each chip were log2 transformed and all data from the sample of one time point were at ASPET Journals on September 25, 2021 quantile normalized using the method described by Amaratunga and Cabrera (Amaratunga and Cabrera, 2001). Following the group-wise quantile normalization, a second quantile normalization was carried out across the data of all samples. Basically this alignment sets the average intensity of one array to the average measured across all arrays, compensating for array to array variations in hybridization, washing and staining, ultimately allowing a reasonable comparison between arrays.

Correspondence factor analysis and spectral map analysis. Correspondence factor analysis and spectral map analysis are special cases of multivariate projection methods that help to reduce the complexity (dimensions) of highly dimensional data (n genes versus p samples). In essence these so called unsupervised methods allow the reduction of the complexity of large microarray datasets and provide means to visually inspect and thereby identify clusters of genes and/or subjects in the data. Typically, projected data are displayed in a biplot combining genes and samples in the same plot. Multivariate projection methods are based on

8 Molecular Pharmacology Fast Forward. Published on July 22, 2004 as DOI: 10.1124/mol.104.000950 This article has not been copyedited and formatted. The final version may differ from this version.

MOLPHARM/2004/000950 a derived space with k orthogonal axes. These k axes are linear combinations of the p original measurements (intensities) on the n genes. The axes are constructed in such a way that the first axis lies in the direction (in the multivariate data space) with the largest variability and the last axis in the direction with the smallest variability. In conventional principal component analysis the first axis (principle component) that maximizes the variability is often related to the size of the intensities. Clusters that can be identified based on the first axis will simply differ in absolute size of the intensities. Correspondence factor analysis (CFA) was originally Downloaded from developed for contingency tables and in a sense decomposes the chi-square statistic.

Therefore, distances between objects in CFA have a chi-square distribution. In spectral map analysis (SPM) of log-transformed data, the distances are proportional to ratios of genes or molpharm.aspetjournals.org samples. In microarray data, we are mainly interested in contrasts and not in simple high or low intensities. For that reason both CFA and SPM have the appropriate properties (double closure for CFA and double centering for SPM) that remove the size component from the data. CFA and SPM will look for contrasts in intensities between genes without the nuisance at ASPET Journals on September 25, 2021 effect of the absolute values of the intensities. Microarray data tend to be more reliable with increasing intensity. Therefore, re-introduction of the size component via variable weighting proportional to the mean intensities of genes and samples is required. A biplot (Chapman et al., 2002) created by the first two axis displays the maximal separation of both the genes and the samples. Coinciding clusters of samples and genes on the biplot indicate the genes

(signatures) that are responsible for the separation of the samples. Genes that are located in the general direction of a sample on the biplot should be looked at as potential signatures for the separation of that sample versus the others. Since, in micro-array data there is a large difference in row- and column-dimension, an asymmetric factor scaling is recommended in constructing the biplot. This operation pulls the genes away from the center of the biplot, while leaving the samples at their original places (Wouters et al., 2003). Both methods were carried out in R (version 1.6.1) using the functions described by Wouters et al.

(http://users.pandora.be/luc.wouters/spm.htm). R is an open source implementation of the statistical programming language S (http://www.r-project.org).

9 Molecular Pharmacology Fast Forward. Published on July 22, 2004 as DOI: 10.1124/mol.104.000950 This article has not been copyedited and formatted. The final version may differ from this version.

MOLPHARM/2004/000950 k-means clustering, hierarchical clustering and fold change. Additional analysis based on k-means clustering, hierarchical clustering and fold change criteria was carried out using the

OmniViz program (OmniViz, Maynard, MA). Gene expression fold differences for CRF,

R121919 and CRF+R121919 treatments were calculated at each time point. For those calculations signals at corresponding time points in DMSO treated samples were used as controls to calculate ratios. K-means clustering (By magnitude and shape; 79 clusters) was done on all log2 transformed ratios. Using the numeric query tool genes were selected which Downloaded from were two fold up or down-regulated in any of the experiments. 975 genes out of 6393 showed a two fold change in expression level in at least one of the experiments. 846 genes were selected which were differentially expressed and present in at least one of the experiments. molpharm.aspetjournals.org For those 846 genes a subset view was created and hierarchical clustering (By magnitude and shape; 29 clusters) was done on log2 transformed ratios. Using the OmniViz Treescape view

(see figure 3), 88 genes which showed a difference in expression after treatment with CRF compared to treatment with the antagonist, were selected (see supplementary table 1). at ASPET Journals on September 25, 2021 Quantitative RT-PCR. Microarray data were confirmed using real time PCR analysis. First strand cDNA synthesis was performed on 0.5µg total RNA using random hexamer primers and SuperscriptII RT (Invitrogen). Quantitative PCR was performed on a ABIPrism 7700 cycler (Applied Biosystems, Foster City, CA) using a Taqman PCR kit (Applied Biosystems).

Serial dilutions of cDNA were used to generate standard curves of threshold cycles versus the logarithms of concentration for ß-actin and the genes of interest (see table 1 for sequences of primers (Eurogentec, Seraing, Belgium)). Other quantitative assays were obtained from

Applied Biosystems (Nurr1, Mm00443056_m1; Nur77, Mm00439358_m1; Per1,

Mm00501813_m1; Atf3, Mm00476032_m1; Fyn, Mm00433373_m1; Snk,

Mm00446917_m1; Pim3, Mm00446876_m1; Ptprn, Mm00436138_m1; Dusp1,

Mm00457274_g1). A linear regression line calculated from the standard curves allowed the determination of transcript levels in RNA samples from the different time points. RT-PCR for

CRFR1 and CRFR2 was performed using Amplitaq (Applied Biosystems) on a 9600 cycler

(Applied Biosystems) with 35 cycles and annealing temperature of 55°C.

10 Molecular Pharmacology Fast Forward. Published on July 22, 2004 as DOI: 10.1124/mol.104.000950 This article has not been copyedited and formatted. The final version may differ from this version.

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Results c-fos expression after CRF exposure. Transcriptional response to continuous CRF exposure was studied in the CRFR1-expressing murine AtT-20 pituitary corticotroph-derived adenoma cell line in a time course ranging from 0.5 to 24 hours. Whereas CRFR1 was readily detectable by RT-PCR, CRFR2 expression could not be discerned in AtT-20 cells (figure 1).

In order to identify CRFR1 specific responses, cells were exposed to CRF either in the presence or absence of the specific CRFR1 antagonist R121919 (Heinrichs et al., 2002). To Downloaded from this end cells were incubated in fresh culture medium containing either a maximal stimulatory concentration of 1µM CRF (Xiong et al., 1995), 1µM CRF in the presence of 1µM of

R121919 and 1µM R121919 alone. Maximal stimulatory concentrations were used in order molpharm.aspetjournals.org to enhance differences between CRF and antagonist treated samples. Transcriptional responses were followed over time, starting from 30 minutes until 24h after administration. In order to assess treatment efficacy, c-fos mRNA levels were determined by quantitative PCR on RNA from the different treatments and time points before array experiments were carried at ASPET Journals on September 25, 2021 out. In agreement with previous reports, exposure to CRF elicited a transient surge in c-fos transcription, with levels already going down after 30 minutes to 1h (see figure 2) (Boutillier et al., 1991). This response was almost completely suppressed in the presence of R121919.

Interestingly, 0.1% DMSO induced c-fos expression, however levels were between 5 to 10 times lower compared to CRF induced expression.

Graphical exploration of array data using projection methods. All time points were analyzed on oligonucleotide microarrays interrogating expression levels of approximately

12000 mouse genes and ESTs. Overall analysis of the expression profiles at the different time points and different treatments was performed using multivariate projection methods such as spectral map analysis (SPM) and correspondence factor analysis (CFA). In the SPM biplot showing the first two principal components, samples seemed to be clustered more according to time point (see figure 3A). This is exemplified by the genes that cluster with the samples.

The gene with identifier 95632_f_at (mevalonate kinase) clustered together with samples

11 Molecular Pharmacology Fast Forward. Published on July 22, 2004 as DOI: 10.1124/mol.104.000950 This article has not been copyedited and formatted. The final version may differ from this version.

MOLPHARM/2004/000950 isolated at 4 and 8 hours. This implies that the gene is responsible for the separation of the 4 and 8 hours samples form the other time points. Indeed high levels of expression of the mevalonate kinase gene (95632_f_at) are observed at 4 and 8 hours after the beginning of the exposure, irrespective of treatment (see left inset figure 3A). At the opposite site of the biplot the RIKEN cDNA 4930429H24 gene with identifier 96494_at, is located together with samples exposed for 0.5, 1 and 24 hours indicating that this gene is high expressed at these time points (see right inset figure 3A). Clearly the first two principal components of SPM Downloaded from indicate progressing time to account for most of the observed changes in gene expression.

When applying CFA, a similar clustering by time point was observed when plotting the first two principal components. However, in contrast to the SPM biplot, CRF treated samples molpharm.aspetjournals.org seemed to be separated from the other treatments at the corresponding time point. This is mainly the case for the early time points (1h until 4h). More striking is the SPM biplot of the first versus third principal component, were all CRF treated samples are separated from other samples (Figure 3B). This points towards an overall difference in expression for CRF treated at ASPET Journals on September 25, 2021 samples compared to the other treatments. After 8 hours of exposure this overall difference is diminishing. Of interest is the observation that R121919 treatment suppresses CRF effects as indicated by the clustering of CRF+R121919 samples with either untreated samples or compound only treated samples. The clear separation of the time and CRF exposure effect was not observed in the CFA, even when looking at all principal components, demonstrating a clear difference between the algorithms. One important feature of both correspondence analysis and spectral map analysis is that they allow the identification of genes that contribute to differences in samples. As indicated in the CFA biplot some genes are clustered together with the CRF treated sample. As shown in the inset in figure 3C the transcription factor JunB

(102362_i_at) is readily upregulated at 0.5 h exposure to CRF with a maximum at 1h. As a consequence this gene clusters together with the CRF 1h treated sample. Similarly cAMP responsive element modulator CREM-ICER (100533_s_at) and cholecystokinin (96055_at) are clustered with the CRF treated samples at respectively 2 and 4 hours of exposure. In this

12 Molecular Pharmacology Fast Forward. Published on July 22, 2004 as DOI: 10.1124/mol.104.000950 This article has not been copyedited and formatted. The final version may differ from this version.

MOLPHARM/2004/000950 way SPM, and to a lesser extent CFA, allowed the identification of genes specifically induced by CRF (see table 2).

Cluster and fold change difference analysis. Other methods to identify genes regulated by

CRF include k-means clustering and fold change difference analysis. Because of the obvious influence of time, expression measurements were analyzed relative to those observed in the corresponding time point in DMSO treated control samples. Regulated genes were defined as those showing a greater than 2-fold change in transcript levels at any one time point. Using Downloaded from the OmniViz Treescape view, 88 genes that showed a difference in expression after treatment with CRF compared to treatment with the antagonist, were selected (see material and methods). 17 out of the 88 genes were “early responders”, showing at least a 2-fold change molpharm.aspetjournals.org already after 30 minutes treatment with CRF. 48 genes were “intermediate responders”, responding between 1 to 2 hours after start of the treatment and 23 genes were “late responders”, showing a response after 2 hours or more (figure 4). These responses were all suppressed by the CRFR1 antagonist R121919 (figure 4). Among the early responders were at ASPET Journals on September 25, 2021 known players in the pathways downstream of the CRFR1 such as the transcription factors

Nurr1, Nurr77, Jun-B, validating the assay. Of interest is the observation that 50 of the 88 genes that were identified using this arbitrary fold change criterion were also identified in the unsupervised spectral map and correspondence factor analysis. A direct comparison of the genes identified by the different methods is shown in table 2.

Genes induced by CRF exposure. Remarkable novel players identified using methods outlined above include transcription factors (e.g. hairy/enhancer-of-split related 1 (Hey1), nuclear factor regulated by interleukin 3 (Nfil3), cAMP responsive element modulator (Crem) and prostate specific ets transcription factor (Pse)), receptor and channel regulators (e.g. Ras- related GTP-binding protein (Gem) and receptor (calcitonin) activity modifying protein 3

(Ramp3)), secreted peptides (e.g. calcitonin, cholecystokinin) and proteins involved in intracellular signaling (e.g. regulator of G-protein signaling 2 (Rgs2), cAMP specific phosphodiesterase 4B (Pde4b) and the regulatory subunit phosphatidylinositol 3-kinase, p85).

Other regulated genes comprise Period homolog Per1, fibroblast growth factor receptor 2

13 Molecular Pharmacology Fast Forward. Published on July 22, 2004 as DOI: 10.1124/mol.104.000950 This article has not been copyedited and formatted. The final version may differ from this version.

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(Fgfr2), serum/glucocorticoid regulated kinase and serum-inducible kinase (figure 5).

Intriguingly, all responders identified according to above mentioned criteria were up regulated after exposure to CRF. This induction was transient and nearly all of the induced genes returned to baseline after 4 to 8 hr. Many of the induced transcripts encode proteins that would exert a negative feedback on the CRFR1 signaling (e.g. Pde4, Rgs2, CREM, etc…), possibly contributing to the transient nature of the induction. In addition to this negative feedback, other mechanism such as desensitization of CRFR1 through phosphorylation and Downloaded from internalization contribute to the transient nature of transcriptional induction (Hauger et al.,

2000;Dautzenberg et al., 2002). In this respect it is of interest to note that challenge with CRF quickly downregulates CRFR1 mRNA in rat pituitary cells (Pozzoli et al., 1996). Although molpharm.aspetjournals.org array experiments did not reveal obvious alterations in CRFR1 mRNA, quantitative PCR showed an increase in CRFR1 mRNA in AtT-20 cells exposed to 1µM CRF after 1 (3.4 fold) and 2 (2.2 fold) hours of exposure (figure 6). Of interest is the observation that even in the presence of R121919 an upregulation of CRFR1 mRNA is observed although less pronounced at ASPET Journals on September 25, 2021 and with some delay to the response in CRF treated samples.

Quantitative PCR. Confirmation of microarray data was carried out using quantitative PCR on the same samples used for hybridization experiments and furthermore on an independent experiment. Levels of regulation and the time course identified by microarray experiments were in agreement with those observed by quantitative PCR. For those genes that have been tested, levels of induction compared to the untreated sample are indicated in figure 5. A comparison between the time course identified by microarray and quantitative PCR is shown in figure 7, demonstrating the regulation of Rgs2.

14 Molecular Pharmacology Fast Forward. Published on July 22, 2004 as DOI: 10.1124/mol.104.000950 This article has not been copyedited and formatted. The final version may differ from this version.

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Discussion

To identify transcriptional pathways downstream of the CRFR1 we applied oligonucleotide microarray analysis on AtT-20 exposed to CRF and the CRFR1 specific antagonist R121919.

A transient induction of negative regulators of CRFR1 signaling was observed. Several previously unknown CRF inducible genes were identified such as Rgs2, Pde4b and Crem.

The use of microarray data to explore the mechanism of action of a compound requires efficient analysis tools that allow rapid identification of specific signatures in gene expression Downloaded from across different samples. Projection methods allow this type of graphical exploration by reducing complex multidimensional data to a two-dimensional subspace, in which samples and genes are plotted together such as principal component analysis (PCA) and molpharm.aspetjournals.org correspondence factor analysis (CFA) (Alter et al., 2000;Hastie et al., 2000;Holter et al.,

2000;Fellenberg et al., 2001). Recently spectral map analysis (SPM) was introduced as an alternative unsupervised multivariate projection method. Weighted SPM outperforms PCA, and is at least as powerful as CFA, in finding clusters in the samples, as well as identifying at ASPET Journals on September 25, 2021 genes related to these clusters (Wouters et al., 2003). When applying SPM and CFA to our dataset we identified exposure time to treatment as important discriminator between the different samples. Synchronization of cell cycle in these cultures induced by addition of new medium and serum could possibly account for this phenomenon although accumulation of metabolites and progressing cell culture conditions are additional contributing factors.

Besides time, SPM identified an effect of CRF treatment in the early time points. These effects of CRF are in agreement with activation of several second messenger such as cAMP and Ca2+ upon stimulation with CRF. Moreover all of these effects were blocked by the

CRFR1 specific antagonist R121919. Some of the transcriptional responses can be explained by the phosphorylation of CREB and the subsequent transcription of genes downstream of cAMP responsive elements. These elements have been found for example in the promoters of

Per1, Nurr1, Crem-icer, c-fos. Furthermore the kinetic profile of the induction of these genes corresponds with the observed maximal transcription rate by CREB after 0.5 hr of initial

15 Molecular Pharmacology Fast Forward. Published on July 22, 2004 as DOI: 10.1124/mol.104.000950 This article has not been copyedited and formatted. The final version may differ from this version.

MOLPHARM/2004/000950 cAMP formation. The induction of Crem-icer constitutes a negative feedback mechanism in attenuating transcriptional response to cAMP (Lamas et al., 1997). Of interest is the reported induction of Crem-icer in response to acute stress in the intermediate lobe of the pituitary gland. Mice deficient for Crem-icer show a chronic increase of beta-endorphin levels suggesting that Crem-icer induction may be involved in the modulation of gene expression in response to stress (Mazzucchelli and Sassone-Corsi, 1999). Our results suggest that Crem-icer is directly involved in the modulation of CRF signaling and as a result ablation of Crem-icer Downloaded from could lead to an altered response to stress signals.

Another novel putative negative feedback regulator of CRF signaling is Rgs2. We identified two single CRE motifs in the promoter of the human RGS2 gene, providing a possible molpharm.aspetjournals.org explanation for the early response behavior of this gene upon stimulation with CRF. In support of our findings is a recent report showing that both phosphoinositide signaling and cAMP induce a rapid and transient increase in Rgs2 mRNA in human astrocytoma and neuroblastoma cells (Zmijewski et al., 2001). The Rgs2 protein is a selective inhibitor of Gqα at ASPET Journals on September 25, 2021 function (Heximer et al., 1997). It has been shown that Rgs2 reduces odorant-elicited cAMP production, not by acting on Gα but by directly inhibiting the activity of adenyl cyclase type

III (Sinnarajah et al., 2001). Although Rgs2 was originally identified as an immediate early response gene in activated T lymphocytes, studies in Rgs2 deficient mice indicate that it also plays a role in the modulation of stress related behavior as these mice show increased anxiety and aggression (Oliveira-Dos-Santos et al., 2000).

The induction of the cAMP specific phosphodiesterase 4B gene (Pde4b), encoding an enzyme responsible for hydrolysis of cAMP (particularly in nerve and immune cells) can also be categorized as negative feedback, directly attenuating the cAMP signal.

Another important second messenger generated upon stimulation with CRF is Ca2+. It has been shown that CRF triggers a steady-state depolarization stimulated extracellular Ca2+ entry via voltage-gated Ca2+ channels and raises intracellular Ca2+ concentration through release from inositol 1,4,5-triphosphate (InsP3) sensitive Ca2+ pools (Tse and Lee, 2000). This leads

16 Molecular Pharmacology Fast Forward. Published on July 22, 2004 as DOI: 10.1124/mol.104.000950 This article has not been copyedited and formatted. The final version may differ from this version.

MOLPHARM/2004/000950 to the secretion of ACTH. Wortmannin has been shown to inhibit both calcium-independent and -stimulated secretion from permeabilized AtT-20 cells, indicating a role for phosphatidylinositol-3 kinase (PI-3K) in determining the size of the readily releasable pool of

ACTH (Wilson and Guild, 2001). The p85 regulatory alpha subunit of PI-3K is upregulated in

CRF treated AtT-20 cells,. This upregulation of the regulatory unit might constitute an attenuation of PI-3K activity. In addition to its role as second messenger, intracellular Ca2+ has also been shown to play a critical role in regulating gene expression. Of interest is the Downloaded from regulation of NFIL3/E4BP4 by calcineurin/NFAT and CaM kinase signaling (Nishimura and

Tanaka, 2001), accounting for an increase in NFIL3 mRNa levels upon CRF treatment. In B lymphocytes expression of NFIL3 is induced by interleukin 3 through both the Raf-mitogen- molpharm.aspetjournals.org activated protein kinase and phosphatidylinositol 3-kinase pathways. In this cell type NFIL3 inhibits apoptosis in synergy with Bcl-xL dependent pathways (Kuribara et al., 1999). Our data suggest a role for NFIL3 in prevention of apoptosis in AtT-20 cells, further supporting a cytoprotective effect of CRF by suppression of pro-apoptotic pathways (Radulovic et al., at ASPET Journals on September 25, 2021 2003).

CRF is the most efficacious ACTH secretagogue. Several other prepropeptides mRNAs were found to be upregulated after CRF administration such as cholecystokinin (CCK) and calcitonin (CT). Of interest in this respect is the upregulation of RAMP3. RAMPs control the transport and glycosylation of the calcitonin receptor-like receptor (CRLR). In the case of

RAMP3, it has been shown that together with CRLR it generates an adrenomedullin (ADM) receptor (McLatchie et al., 1998). Upregulation of this gene might play a role in regulating the responsiveness of AtT-20 cells to ADM after CRF exposure. In addition regulation of

RAMP3 could influence responses to other extracellular stimuli such as CRF, as it is known that RAMPs might regulate other G-coupled receptors for example vasoactive intestinal polypetide receptor and parathyroid receptor (Christopoulos et al., 2003).

Although CCK is secreted by AtT-20 cells, induction of its expression by CRF has not been previously reported (Beinfeld, 1992). Interaction between CCK and CRF has however been intensively studied and demonstrated in panic attacks, depression, anxiety and gastric

17 Molecular Pharmacology Fast Forward. Published on July 22, 2004 as DOI: 10.1124/mol.104.000950 This article has not been copyedited and formatted. The final version may differ from this version.

MOLPHARM/2004/000950 emptying (Coskun et al., 1997;Geracioti, Jr. et al., 1999;Kellner et al., 2000). Most of these experiments point towards a role for CRF in mediating the central effects of CCK. Our data indicate that CRF in addition might function as a CCK secretagogue.

In agreement with previous reports we could find a upregulation of CRFR1 mRNA after prolonged CRFR1 exposure (Iredale et al., 1996). It is tempting to speculate that this upregulation compensates for the loss of receptor at the surface after internalization and restores responsiveness of the cells. An interesting observation is the similar upregulation Downloaded from with a shift in time in the cells treated with CRF in the presence of the antagonist. This suggests that also these receptor complexes are internalized.

In conclusion, we have unraveled part of the corticotropin-releasing hormone receptor-1 molpharm.aspetjournals.org activated gene network and have identified several novel targets of this signaling cascade. In addition we have demonstrated the power of spectral map analysis for microarray data exploration. at ASPET Journals on September 25, 2021

18 Molecular Pharmacology Fast Forward. Published on July 22, 2004 as DOI: 10.1124/mol.104.000950 This article has not been copyedited and formatted. The final version may differ from this version.

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at ASPET Journals on September 25, 2021

22 Molecular Pharmacology Fast Forward. Published on July 22, 2004 as DOI: 10.1124/mol.104.000950 This article has not been copyedited and formatted. The final version may differ from this version.

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Footnotes b) Reprint requests: Pieter Peeters, Johnson & Johnson Pharmaceutical Research and

Development, a division of Janssen Pharmaceutica N.V., Turnhoutseweg 30, B-2430 Beerse,

Belgium Downloaded from molpharm.aspetjournals.org at ASPET Journals on September 25, 2021

23 Molecular Pharmacology Fast Forward. Published on July 22, 2004 as DOI: 10.1124/mol.104.000950 This article has not been copyedited and formatted. The final version may differ from this version.

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Legends for Figures

Figure 1

Title: expression CRFR1 and CRFR2 in AtT-20 cells.

Legend: RT-PCR reaction were performed on two isolates of AtT-20 cells, mouse heart and mouse brain RNA with primers specific for mouse CRFR1 and CRFR2. Both receptors are expressed in brain whereas only CRFR2 is expressed in heart. As a control for RNA quality Downloaded from primers for ß-actin were used.

Figure 2 molpharm.aspetjournals.org

Title: normalized c-fos expression after CRF treatment.

Legend: c-fos mRNA levels as assessed by quantitative RT-PCR normalized against ß-actin mRNA levels in AtT-20 cells continuously exposed to DMSO, 1µM CRF, 1µM

CRF+R121919 or 1µM R121919 in a time course ranging from 30 minutes to 24h. at ASPET Journals on September 25, 2021

Figure 3

Title: Spectral Map and Correspondence Analysis of Microarray data.

Legend: A – First two principal components (PC) of the weighted Spectral map analysis

(SPM) applied on normalized microarray data for all time points and treatments. On the spectral map squares depict different samples whereas circles depict genes (size of circle correspond to intensity). Distances between squares are a measure for similarity between samples. A positive association of a gene with a given sample (i.e. an upregulation of that gene in that particular sample) results in the positioning of the gene and sample on a common line through the centroid (depicted by a cross). Genes contributing significantly (measured by their distance form the centroid) to difference between samples are annotated with their

Affymetrix identifier (www.affymetrix.com/analysis/netaffx) (see table 2 for annotation).

First two PCs identified time as the major discriminator between the samples. Inserted heat

24 Molecular Pharmacology Fast Forward. Published on July 22, 2004 as DOI: 10.1124/mol.104.000950 This article has not been copyedited and formatted. The final version may differ from this version.

MOLPHARM/2004/000950 maps (insets) show representative genes (indicated in bold in biplot) that are maximally induced either after 4 and 8 h or at 0.5, 1 and 24h, corresponding to their position on the biplot. B – biplot of first and third PCs. Third PC identifies the specific CRF effect on AtT-20 cells over time. C – Summary views on the first three dimensions of spectral map, showing how this technique identified time and CRF effects in the microarray dataset. D -

Correspondence factor analysis (CFA) applied on the same data as in A. The interpretation of the CFA output is similar to that of SPM. In addition to time, CFA identified the effect of Downloaded from CRF exposure as most prominent in the early time points (1-4h). Inserted heat maps show representative genes that are maximally induced either after 1, 2 or 4 h of exposure to CRF, corresponding to their position on the map. molpharm.aspetjournals.org

Figure 4

Title: Heat map of selected genes affected by CRF exposure.

Legend: A heat map depicting 88 genes that are changed upon CRF treatment as identified by at ASPET Journals on September 25, 2021 a combination of cluster analysis and fold change criteria (for list of 88 genes see supplementary table 1). Values were calculated by dividing the intensity of each sample by the intensity of the DMSO sample at the corresponding time point. These calculated ratio are converted into a colour ramp on a log2 scale. In this way the different timing of induction of expression becomes apparent. Genes showing a 2-fold change after 30 minutes of treatment with CRF were called “early responders”, “intermediate responders” show a change after 1 to

2 hours of treatment and “late responders” show a response after 2 hours or more.

Figure 5

Title: Overview of regulated pathways.

Legend: Overview of a selection of genes induced by CRF grouped by pathway or function.

Values were calculated by dividing the intensity of each sample by the intensity of the DMSO sample at the corresponding time point. These calculated ratio are converted into a color ramp on a log2 scale and depicted in a heat map. Next to the heat maps the maximal fold change

25 Molecular Pharmacology Fast Forward. Published on July 22, 2004 as DOI: 10.1124/mol.104.000950 This article has not been copyedited and formatted. The final version may differ from this version.

MOLPHARM/2004/000950 over all timepoints as detected by quantitative PCR is shown for each gene tested. These PCR data were obtained on a repeated experiment. For an overview of all quantitative PCR data see supplementary table 2. At the right Affymetrix identifiers for each gene are depicted.

(ND, not done)

Figure 6

Title: Induction of CRFR1 mRNA by CRF in AtT-20 cells. Downloaded from Legend: Quantitative PCR results with CRFR1 specific primers. Induction is calculated in comparison to levels observed in AtT-20 cells before any treatment.

molpharm.aspetjournals.org Figure 7

Title: Induction of Rgs2 mRNA by CRF in AtT-20 cells.

Legend: Induction is calculated in comparison to levels observed in AtT-20 cells before any treatment. On top, array data obtained for Rgs2 are shown. Below, levels of Rgs2 mRNA as at ASPET Journals on September 25, 2021 measured by quantitative RT-PCR on the same samples as used for array experiments and as measured in an independent experiment.

26 Molecular Pharmacology Fast Forward. Published on July 22, 2004 as DOI: 10.1124/mol.104.000950 This article has not been copyedited and formatted. The final version may differ from this version.

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Tables

Table 1

Sequence of oligonucleotides used for quantitative PCR

ß-actin forward 5’-CATCTTGGCCTCACTGTCCAC-3’

ß-actin probe 5’-TGCTTGCTGATCCACATCTGCTGGA-3’

ß-actin reverse 5’-GGGCCGGACTCATCGTACT-3’ c-fos forward 5’-GGGAGGACCTTACCTGTTCGT-3’ Downloaded from c-fos probe 5’-CACCAGGCTGTGGGCCTCAAGG-3’ c-fos reverse 5’-CCAGATGTGGATGCTTGCAA-3’

Crf2 forward 5’-GGGAGAACAGAAGCGCCTG-3’ molpharm.aspetjournals.org

Crf2 probe 5’-AGAAGGGTGAGGATCCCCCAAATCAGAGT-3’

Crf2 reverse 5’-CCCTTGTTTCAATCACTCCCA-3’

Crf1 forward 5’-TTTCTGAACAGTGAGGTCCGC-3’

Crf1 probe 5’-CCGGAAGAGGTGGCGGCGA-3’ at ASPET Journals on September 25, 2021 Crf1 reverse 5’-GGGCTCTGATGGAGTGCTTG-3’

Rgs2 forward 5’-TTGGAAGACCCGTTTGAGCTA-3’

Rgs2 probe 5’-TCTTGCAGAATTCCTCTGCTCCTGGG-3’

Rgs2 reverse 5’-TTTCTTGCCAGTTTTGGGCT-3’

Fgfr2 forward 5’-AGACTTCCATGGGAATGATAGCA-3’

Fgfr2 probe 5’-CCTCTCGTCCGGCAGCTGGC-3’

Fgfr2 reverse 5’-AATGTGTAAGCCGGGCAGAA-3’

Mig6 forward 5’-AATCCTTTGTCCAATACTGTACACACA-3’

Mig6 probe 5’-GAAAAATGCACTGATCTCCGCA-3’

Mig6 reverse 5’-GTATGAACTAAATGAAGGTTAAAACATGCT-3’

Pi3k forward 5’-CCATGGTGCTTGTTAACGCTTT-3’

Pi3k probe 5’-CCCAACTTGTAGCTGGTAAAGCTTCA-3’

Pi3k reverse 5’-CCTGTCTACCTTCTGGTCTCCAA-3’

1 Molecular Pharmacology Fast Forward. Published on July 22, 2004 as DOI: 10.1124/mol.104.000950 This article has not been copyedited and formatted. The final version may differ from this version.

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Crem forward 5’-CTTGCTGATCGTCTGGAGAGTTT-3’

Crem probe 5’-TGCTGATGACCCTCCATTGTGA-3’

Crem reverse 5’-TTAACATTCCTGAGGTTGCAAGAA-3’

Pde4b forward 5’-GCCGTGTGTATGGCTGCAT-3’

Pde4b probe 5’-CAGCCCCCAGGCCACTGTGG-3’

Pde4b reverse 5’-AGGAGGGATAACAGGTGCTGTGT-3’

CCK forward 5’-CCTGGACCCCAGCCATAGA-3’ Downloaded from CCK probe 5’-AGCCCATGTAGTCCCGGTCACTTA-3’

CCK reverse 5’-TGCGCCGGCCAAAA-3’

CT forward 5’-GCTTGGACAGCCCCAGATC-3’ molpharm.aspetjournals.org CT probe 5’-GGTACTCAGATTCCCACACCGCTT-3’

CT reverse 5’-TGTGTGTACGTGCCCAGCAT-3’

Nfil3 forward 5’-GCGAGTTTGAAGGCATGCA-3’

Nfil3 probe 5’-CTCTCTTCACCCGCCGATGCGAT-3’ at ASPET Journals on September 25, 2021 Nfil3 reverse 5’-CCATGTTTCTCCAGGTCAAAATG-3’

Ramp3 forward 5’-TGGCAGACTCGGCTTCTGT-3’

Ramp3 probe 5’-TTTGCTTTGGCCACACCCTACCTGG-3’

Ramp3 reverse 5’-CTGGTCGGGAGGACTTTGG-3’

Sgk forward 5’-TGGACCAATGCCCCAGTT-3’

Sgk probe 5’-TCAGTCAAAGCCGTTGGTGTTTTCATTG-3’

Sgk reverse 5’-GCCCGTTTTATAGGTGACATTTTAA-3’

2 Molecular Pharmacology Fast Forward. Published on July 22, 2004 as DOI: 10.1124/mol.104.000950 This article has not been copyedited and formatted. The final version may differ from this version.

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Table 2

Genes identified on SPM and CFA biplots

SPM PC1vsPC2

Probe Set ID Gene Symbol Gene name

100050_at Idb1 inhibitor of DNA binding 1

102362_i_at Junb Jun-B oncogene

102397_at Cbfa2t3h core-binding factor, runt domain, alpha subunit 2; translocated to, 3 homolog

102410_at Hs3st1 heparan sulfate (glucosamine) 3-O-sulfotransferase 1

103748_at 4933407C03Rik RIKEN cDNA 4933407C03 gene Downloaded from 104588_at 1810073K19Rik RIKEN cDNA 1810073K19 gene

94428_at Ilvbl ilvB (bacterial acetolactate synthase)-like

95632_f_at Mvk mevalonate kinase

96494_at 4930429H24Rik RIKEN cDNA 4930429H24 gene molpharm.aspetjournals.org

96803_at Gbe1 glucan (1,4-alpha-), branching enzyme 1

98478_at Ccng2 cyclin G2

98855_r_at --- weak similarity to melanoma-associated chondroitin sulfate proteoglycan 4

99077_at Thra thyroid hormone receptor alpha

SPM PC1vsPC3 at ASPET Journals on September 25, 2021

Probe Set ID Gene Symbol Gene name

100533_s_at Crem cAMP responsive element modulator

102362_i_at Junb Jun-B oncogene

102410_at Hs3st1 heparan sulfate (glucosamine) 3-O-sulfotransferase 1

102907_at C820004H04Rik RIKEN cDNA C820004H04 gene

104588_at 1810073K19Rik RIKEN cDNA 1810073K19 gene

104712_at Myc myelocytomatosis oncogene

92205_at --- moderate similarity to insulin receptor substrate 2

94428_at Ilvbl ilvB (bacterial acetolactate synthase)-like

95632_f_at Mvk mevalonate kinase

96055_at Cck cholecystokinin

96326_at Tat tyrosine aminotransferase

96494_at 4930429H24Rik RIKEN cDNA 4930429H24 gene

97844_at Rgs2 regulator of G-protein signaling 2

3 Molecular Pharmacology Fast Forward. Published on July 22, 2004 as DOI: 10.1124/mol.104.000950 This article has not been copyedited and formatted. The final version may differ from this version.

MOLPHARM/2004/000950

CFA

Probe Set ID Gene Symbol Gene name

100533_s_at Crem cAMP responsive element modulator

102362_i_at Junb Jun-B oncogene

102363_r_at Junb Jun-B oncogene

102371_at Nr4a1 nuclear receptor subfamily 4, group A, member 1

104712_at Myc myelocytomatosis oncogene

92248_at Nr4a2 nuclear receptor subfamily 4, group A, member 2

93090_at Fgfr2 fibroblast growth factor receptor 2

93091_s_at Fgfr2 fibroblast growth factor receptor 2 Downloaded from

93974_at 1300002F13Rik RIKEN cDNA 1300002F13 gene

96055_at Cck cholecystokinin

96326_at Tat tyrosine aminotransferase

97844_at Rgs2 regulator of G-protein signaling 2 molpharm.aspetjournals.org

99329_at Abcc1 ATP-binding cassette, sub-family C at ASPET Journals on September 25, 2021

4 Molecular Pharmacology Fast Forward. Published on July 22, 2004 as DOI: 10.1124/mol.104.000950 This article has not been copyedited and formatted. The final version may differ from this version. MOLPHARM/2004/000950 Downloaded from 78 bp 76 bp 72 bp

molpharm.aspetjournals.org

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T t A CRFR2 CRFR1 ß-actin Peeters 1 Figure al., et Peeters et al., Figure 2 MOLPHARM/2004/000950 Molecular PharmacologyFastForward.PublishedonJuly22,2004asDOI:10.1124/mol.104.000950 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion.

35 0 30 DMSO CRF 25 CRF+R121919 20 R121919

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0 0.50 1.00 2.00 4.00 8.00 24.00

time [h]

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SPM - PC1 vs. PC2 Molecular PharmacologyFastForward.PublishedonJuly22,2004asDOI:10.1124/mol.104.000950 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion.

24.DMSO

24.CRF+R121919 24.CRF 24.R121919 98478_at 08.CRF+R121919 08.R121919 08.DMSO 102410_at 08.CRF T0 96494_at 96803_at 98855_r_at 0.5.R121919 0.5.DMSO 99077_at 0.5.CRF 94428_at 95632_f_at 01.R121919 0.5.CRF+R121919 PC2 15 15 % PC2 104588_at 04.CRF+R121919 01.DMSO 04.R121919 02.CRF+R121919 01.CRF 04.CRF 04.DMSO 02.DMSO 102362_i_at 01.CRF+R121919 95632_f_at 96494_at 103748_at 0,5 1 2 4 8 24 h 02.CRF 02.R121919 0,5124824 h DMSO DMSO CRF 102397_at 100050_at CRF R R CRF+R CRF+R

1234567 1234567

PC1 25 %

Closure = none, Center = double, Norm. = global, Scale = uvc, RW = mean, CW = constant

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100533_s_at 104712_at 96326_at 97844_at 96055_at 02.CRF 102907_at 102362_i_at 04.CRF 08.CRF 01.CRF 92205_at 24.DMSO 0.5.CRF

95632_f_at

08.CRF+R121919 24.CRF+R121919 PC3 9 % PC3 0.5.DMSO 96494_at 01.CRF+R121919 08.DMSO 02.CRF+R121919 0.5.R121919 04.CRF+R121919 0.5.CRF+R121919 24.CRF 94428_at T0 08.R121919 02.DMSO 102410_at 01.DMSO 04.R121919 24.R121919 04.DMSO 01.R121919

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24h T0 T0 DMSO DMSO CRF CRF 8h R R CRF & R CRF & R

0h CRF PC3 9 % 9 PC3 PC2 15 % PC2 15

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CFA

102362_i_at

02.CRF Molecular PharmacologyFastForward.PublishedonJuly22,2004asDOI:10.1124/mol.104.000950

0,5 1 2 4 8 24 h This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. DMSO CRF 100533_s_at R CRF+R

12345678910 96326_at 01.CRF 97844_at 104712_at 93091_s_at 04.CRF 99392_at 93974_at 93090_at 102362_i_at 92248_at 102371_at 100533_s_at 96055_at DMSO 102363_r_at 02.R121919 CRF 01.CRF+R121919 02.CRF+R121919

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Closure = double, Center = double, Norm. = global, Scale = uvc, RW = mean, CW = mean

Downloaded from from Downloaded molpharm.aspetjournals.org at ASPET Journals on September 25, 2021 25, September on Journals ASPET at Peeters et al., Figure 4 Figure al., et Peeters

0.5h CRF

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2h CRF

4h CRF

8h CRF

24h CRF

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molpharm.aspetjournals.org 24h R121919

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This article has not been copyedited and formatted. The final version may differ from this version. this from differ may version final The formatted. and copyedited been not has article This Molecular Pharmacology Fast Forward. Published on July 22, 2004 as DOI: 10.1124/mol.104.000950 DOI: as 2004 22, July on Published Forward. Fast Pharmacology Molecular Peeters et al., Figure 5A MOLPHARM/2004/000950 Molecular PharmacologyFastForward.PublishedonJuly22,2004asDOI:10.1124/mol.104.000950 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion.

Transcription factors 24h CRF 24h RTq 0.5h CRF 0.5h CRF 1h CRF 4h CRF 8h R121919 0.5h 1h R121919 8h R121919 R121919 24h CRF+R121919 0.5h CRF+R121919 1h CRF+R121919 2h CRF+R121919 4h CRF+R121919 8h CRF+R121919 24h 2h CRF 2h 2h R121919 4h R121919

prostate specific ets transcription factor ND 104008_at

nuclear receptor subfamily 4, group A, member 1 7.09 102371_at

nuclear receptor subfamily 4, group A, member 2 2.67 92249_g_at

Jun-B oncogene ND 102362_i_at

nuclear receptor subfamily 4, group A, member 2 2.67 92248_at

period homolog 2.95 93619_at

nuclear factor, interleukin 3 regulated 3.20 102955_at

activating transcription factor 3 2.10 104155_f_at

activating transcription factor 3 2.10 104156_r_at

-4 -3 -2 -1 0 1 2 3 4

Downloaded from from Downloaded molpharm.aspetjournals.org at ASPET Journals on September 25, 2021 25, September on Journals ASPET at Peeters et al., Figure 5B MOLPHARM/2004/000950 Molecular PharmacologyFastForward.PublishedonJuly22,2004asDOI:10.1124/mol.104.000950 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion.

Kinases RTq 0.5h CRF 0.5h CRF 1h CRF 4h CRF 8h CRF 24h R121919 0.5h 1h R121919 8h R121919 R121919 24h CRF+R121919 0.5h CRF+R121919 1h CRF+R121919 2h CRF+R121919 4h CRF+R121919 8h CRF+R121919 24h 2h CRF 2h 2h R121919 4h R121919

fibroblast growth factor receptor 2, Fgfr2 9.71 93090_at

serum/glucocorticoid regulated kinase, Sgk 6.18 97890_at

fibroblast growth factor receptor 2 9.71 93091_s_at

Fyn proto-oncogene, Fyn 1.84 100133_at

serine threonine kinase, Pim3 10.23 96841_at

serum inducible kinase, Snk 3.25 92310_at

-4-3-2-101234 cAMP signaling

phosphodiesterase 4B, cAMP specific 3.99 103610_at

cAMP responsive element modulator 45.52 100533_s_at

regulator of G-protein signaling 2, Rgs2 18.10 97844_at

-4-3-2-101234

Downloaded from from Downloaded molpharm.aspetjournals.org at ASPET Journals on September 25, 2021 25, September on Journals ASPET at Peeters et al., Figure 5C MOLPHARM/2004/000950 Molecular PharmacologyFastForward.PublishedonJuly22,2004asDOI:10.1124/mol.104.000950 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion.

Secreted peptides RTq 0.5h CRF 0.5h CRF 1h CRF 4h CRF 8h CRF 24h CRF+R121919 24h 2h CRF 2h R121919 0.5h 1h R121919 8h R121919 R121919 24h CRF+R121919 0.5h CRF+R121919 1h CRF+R121919 2h CRF+R121919 4h CRF+R121919 8h 2h R121919 4h R121919

Calcitonin, Ct 36.33 92533_at

Cholecystokinin, Cck 28.94 96055_at

-4-3-2-101234

Inositol signaling

phosphatidylinositol 3-kinase, regulatory subunit 4.48 94977_at

-4-3-2-101234

Receptor and channel regulators

receptor (calcitonin) activity modifying protein 3 14.44 92368_at

-4-3-2-101234

Downloaded from from Downloaded molpharm.aspetjournals.org at ASPET Journals on September 25, 2021 25, September on Journals ASPET at Peeters et al., Figure 5D MOLPHARM/2004/000950 Molecular PharmacologyFastForward.PublishedonJuly22,2004asDOI:10.1124/mol.104.000950 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion.

Phosphatases 0.5h CRF 0.5h CRF 1h CRF 4h CRF 8h CRF 24h R121919 24h CRF+R121919 0.5h CRF+R121919 1h CRF+R121919 2h CRF+R121919 4h CRF+R121919 8h CRF+R121919 24h RTq 2h CRF 2h R121919 0.5h 1h R121919 8h R121919 2h R121919 4h R121919

protein tyrosine phosphatase, receptor-type, N 39.16 104422_at

dual-specificity phosphatase 1, Dusp1 9.99 104598_at

-4-3-2-101234 Unknowns

MGC:37790 IMAGE:5097591 ND 96326_at

RIKEN cDNA 1300002F13 gene (MIG-6) 3.17 93974_at

tumor necrosis factor, alpha-induced protein 3 ND 99392_at

-4-3-2-101234

Downloaded from from Downloaded molpharm.aspetjournals.org at ASPET Journals on September 25, 2021 25, September on Journals ASPET at Peeters et al., Figure 6 MOLPHARM/2004/000950 Molecular PharmacologyFastForward.PublishedonJuly22,2004asDOI:10.1124/mol.104.000950 4 This articlehasnotbeencopyeditedandformatted.Thefinalversionmaydifferfromthisversion. DMSO 3.5 CRF CRF+R121919 0 3

2.5

2

1.5

1 fold change compared to T compared change fold

0.5

0 0.512468

time [h]

Downloaded from from Downloaded molpharm.aspetjournals.org at ASPET Journals on September 25, 2021 25, September on Journals ASPET at Molecular Pharmacology Fast Forward. Published on July 22, 2004 as DOI: 10.1124/mol.104.000950 Peeters et al., FigureThis article 7 has not been copyedited and formatted. The final version may differMOLPHARM/2004/000950 from this version.

array data 12 DMSO ) 10 0 R121919 8 CRF+R121919 6 CRF 4 fold change fold (compared to T 2 0

0.5 124824 Downloaded from molpharm.aspetjournals.org RTq data 35 30 ) 0 25 20

15 at ASPET Journals on September 25, 2021 10 fold change fold (compared to T 5 0 0.5 1 2 4824

RTq data independent exp. 20 ) 0 16 12 8 fold change fold (compared to T 4 0 0.5 12468 time [h] Supplementary Table 2 from Peeters et al., Mol Pharmacol, doi: 10.1124/mol.104.000950

Overview of quantitative PCR data for selected genes expressed as fold change compared to T0

Nfil3 Ramp3 Cck Ct Fgfr2Mig-6 Pde4b Pi3k Crem-icer Sgk Atf3 Fyn Per1 Snk Pim3 Nr4a2 Ptpn Nr4a1 Dusp1 Rgs2 Crfr1

T0 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 1.00 C+R 0.5 0.96 0.90 1.59 1.41 0.78 0.46 1.24 0.81 1.44 2.20 1.09 0.55 1.11 0.99 1.26 0.72 3.00 1.25 1.64 1.27 0.85 C+R 1 1.11 1.44 2.49 2.57 1.41 0.96 1.31 0.68 2.96 2.80 1.03 0.27 0.90 0.65 1.68 1.04 5.21 2.76 0.76 1.99 0.75 C+R 2 0.88 1.37 3.03 2.81 2.52 1.17 1.54 1.15 2.23 3.81 0.89 0.68 1.27 0.71 1.16 1.37 1.37 1.67 0.26 2.55 2.01 C+R 4 1.06 0.67 2.67 3.41 0.76 1.38 1.24 1.31 1.63 1.65 1.00 0.97 1.95 1.02 1.45 1.98 1.14 1.09 0.55 2.03 1.65 C+R 6 0.95 0.49 2.00 0.99 1.16 1.60 2.16 1.01 1.13 1.65 0.25 0.70 0.58 0.55 0.42 0.96 0.46 0.74 0.38 1.51 0.42 C+R 8 0.77 0.41 1.90 1.64 0.29 0.29 0.61 0.15 1.81 2.07 0.11 0.31 0.63 0.77 0.38 0.57 0.30 0.42 0.28 1.13 1.07 C 0.5 2.31 1.79 4.56 1.13 0.72 1.28 1.90 0.64 13.27 2.43 0.22 0.41 1.14 1.74 7.09 0.93 19.57 2.28 7.34 7.70 0.43 C 1 3.20 4.13 6.91 34.77 2.33 3.80 2.02 1.94 24.35 3.25 2.10 0.54 2.95 3.26 10.23 1.32 39.17 7.10 10.00 18.12 3.40 C 2 1.22 13.57 26.57 36.33 9.71 8.68 3.99 4.48 45.52 6.18 0.34 1.84 1.77 1.94 1.49 2.68 9.41 5.72 1.11 10.54 2.23 C 4 1.28 14.40 23.42 18.52 2.21 6.39 2.41 0.96 7.22 3.37 0.33 0.97 0.68 1.00 0.51 1.40 1.27 1.00 0.41 2.32 0.74 C 6 1.43 8.12 28.49 5.53 1.99 2.46 1.36 0.72 3.54 2.08 0.38 0.24 0.84 0.22 1.24 2.25 2.58 1.26 0.48 2.10 0.91 C 8 1.08 4.29 24.17 9.79 4.28 8.95 3.77 1.29 1.71 1.49 1.58 0.58 1.26 1.15 1.19 2.15 3.09 0.86 0.34 1.64 1.22 D 0.5 0.62 0.48 2.31 1.35 1.21 0.30 0.83 0.60 0.76 2.82 0.57 0.34 0.43 0.61 0.24 0.38 0.48 0.46 0.15 1.00 0.41 D 1 0.42 0.41 1.51 0.32 2.07 0.32 0.69 0.72 0.31 0.99 0.16 0.19 0.48 0.24 0.25 0.43 0.35 0.39 0.12 0.94 0.59 D 2 0.38 0.59 1.34 0.81 1.54 0.23 0.84 0.45 0.66 1.53 0.03 0.48 0.62 0.34 0.41 0.40 0.30 0.47 0.17 1.16 0.66 D 4 0.36 0.41 0.93 0.64 0.80 0.34 0.90 0.60 0.57 0.68 0.89 0.54 0.68 0.62 0.56 0.38 0.24 0.48 0.27 1.00 0.42 D 6 0.42 0.47 1.03 0.59 0.59 0.18 0.96 0.58 0.91 1.17 0.58 0.45 0.93 0.37 1.13 0.76 0.49 0.75 0.31 0.68 0.61 D 8 0.43 0.38 1.29 1.27 0.69 0.25 0.70 0.72 1.00 1.19 0.74 0.23 0.29 0.28 0.54 0.40 0.29 0.35 0.19 0.73 0.36 Max* 3.20 14.40 28.49 36.33 9.71 8.95 3.99 4.48 45.52 6.18 2.10 1.84 2.95 3.26 10.23 2.68 39.17 7.10 10.00 18.12 3.40

*Maximum induction

1 Supplementary Table 1 from Peeters et al., Mol Pharmacol, doi: 10.1124/mol.104.000950

Affymetrix identified by ID SMA CFA OmniViz Gene Title Gene Symbol 100133_at yes yes yes Fyn proto-oncogene Fyn 100533_s_at yes yes yes cAMP responsive element modulator Crem 101196_at yes yes yes paired basic amino acid cleaving system 4 Pace4 101217_at yes yes yes ------101426_at yes yes yes ceramide kinase Cerk 102231_at yes yes yes cAMP responsive element binding protein 3-like 1 Creb3l1 102239_at yes yes yes B-cell leukemia/lymphoma 3 Bcl3 102362_i_at yes yes yes Jun-B oncogene Junb 102363_r_at yes yes yes Jun-B oncogene Junb 102371_at yes yes yes nuclear receptor subfamily 4, group A, member 1 Nr4a1 102955_at yes yes yes nuclear factor, interleukin 3, regulated Nfil3 104008_at yes yes yes SAM pointed domain containing ets transcription factor Spdef 104410_at yes yes yes midnolin Midn 104598_at yes yes yes dual specificity phosphatase 1 Dusp1 104669_at yes yes yes interferon regulatory factor 7 Irf7 92205_at yes yes yes insulin receptor substrate 2 Irs2 92248_at yes yes yes nuclear receptor subfamily 4, group A, member 2 Nr4a2 92249_g_at yes yes yes nuclear receptor subfamily 4, group A, member 2 Nr4a2 92310_at yes yes yes polo-like kinase 2 (Drosophila) Plk2 92368_at yes yes yes receptor (calcitonin) activity modifying protein 3 Ramp3 92406_at yes yes yes CD7 antigen Cd7

1 92423_at yes yes yes par-6 (partitioning defective 6,) homolog alpha (C. elegans) Pard6a 92820_at yes yes yes ubiquitin specific protease 2 Usp2 92821_at yes yes yes ubiquitin specific protease 2 Usp2 92925_at yes yes yes CCAAT/enhancer binding protein (C/EBP), beta Cebpb 93090_at yes yes yes fibroblast growth factor receptor 2 Fgfr2 93091_s_at yes yes yes fibroblast growth factor receptor 2 Fgfr2 93318_at yes yes yes ninjurin 1 Ninj1 93619_at yes yes yes period homolog 1 (Drosophila) Per1 93738_at yes yes yes solute carrier family 2 (facilitated glucose transporter), member 1 Slc2a1 93753_at yes yes yes LPS-induced TN factor Litaf 93974_at yes yes yes RIKEN cDNA 1300002F13 gene 1300002F13Rik 93975_at yes yes yes RIKEN cDNA 1300002F13 gene 1300002F13Rik 94384_at yes yes yes immediate early response 3 Ier3 sema domain, immunoglobulin domain (Ig), transmembrane domain (TM) and short cytoplasmic domain, 95387_f_at yes yes yes Sema4b (semaphorin) 4B 95418_at yes yes yes RAS-like, family 11, member B Rasl11b 95671_at yes yes yes hairy/enhancer-of-split related with YRPW motif 1 Hey1 96055_at yes yes yes cholecystokinin Cck 96326_at yes yes yes tyrosine aminotransferase Tat 96524_at yes yes yes RIKEN cDNA D030068L24 gene D030068L24Rik 96592_at yes yes yes phosphatidylinositol 3-kinase, regulatory subunit, polypeptide 1 (p85 alpha) Pik3r1 96841_at yes yes yes proviral integration site 3 Pim3 97386_at yes yes yes RIKEN cDNA 1110032O19 gene 1110032O19Rik 97844_at yes yes yes regulator of G-protein signaling 2 Rgs2 97890_at yes yes yes serum/glucocorticoid regulated kinase Sgk 98569_at yes yes yes solute carrier family 25 (mitochondrial carrier, phosphate carrier), member 25 Slc25a25 98868_at yes yes yes B-cell leukemia/lymphoma 2 Bcl2

2 99392_at yes yes yes tumor necrosis factor, alpha-induced protein 3 Tnfaip3 99622_at yes yes yes Kruppel-like factor 4 (gut) Klf4 99994_at yes yes yes cell death-inducing DNA fragmentation factor, alpha subunit-like effector A Cidea 100365_at yes yes no synaptotagmin 7 Syt7 102907_at yes yes no pleckstrin homology-like domain, family B, member 2 Phldb2 104701_at yes yes no basic helix-loop-helix domain containing, class B2 Bhlhb2 104712_at yes yes no myelocytomatosis oncogene Myc 92833_at yes yes no histidine ammonia lyase Hal 93694_at yes yes no period homolog 2 (Drosophila) Per2 94051_at yes yes no EGF-like repeats and discordin I-like domains 3 Edil3 95552_at yes yes no deiodinase, iodothyronine, type I Dio1 100386_at yes no yes guanine nucleotide binding protein, alpha z subunit Gnaz 100511_at yes no yes RIKEN cDNA 6330406L22 gene 6330406L22Rik 100530_at yes no yes ral guanine nucleotide dissociation stimulator Ralgds 100716_at yes no yes hyaluronan synthase1 Has1 101437_at yes no yes serine/threonine kinase 2 Stk2 101651_at yes no yes ciliary neurotrophic factor receptor Cntfr 101940_at yes no yes sphingomyelin phosphodiesterase 2, neutral Smpd2 102292_at yes no yes growth arrest and DNA-damage-inducible 45 alpha Gadd45a 102299_at yes no yes protein kinase C, alpha Prkca 102631_at yes no yes Bloom syndrome homolog (human) Blm 102661_at yes no yes early growth response 2 Egr2 103095_at yes no yes growth factor receptor bound protein 7 Grb7 103420_at yes no yes emerin Emd 103610_at yes no yes phosphodiesterase 4B, cAMP specific Pde4b 104389_at yes no yes RIKEN cDNA 1700017B05 gene 1700017B05Rik

3 104422_at yes no yes protein tyrosine phosphatase, receptor-type, N Ptprn 104699_at yes no yes phosphoinositide-3-kinase, class 3 Pik3c3 92232_at yes no yes suppressor of cytokine signaling 3 Socs3 93478_at yes no yes ES cells cDNA, RIKEN full-length enriched library, clone:C330022M23 product:unclassifiable, full insert sequence --- 93860_i_at yes no yes ------93861_f_at yes no yes ------94803_at yes no yes pre B-cell leukemia transcription factor 1 Pbx1 94815_at yes no yes 2,3-bisphosphoglycerate mutase Bpgm 94902_at yes no yes superoxide dismutase 3, extracellular Sod3 94977_at yes no yes inositol 1,4,5-triphosphate receptor 1 Itpr1 95466_at yes no yes coactosin-like 1 (Dictyostelium) Cotl1 96871_at yes no yes RIKEN cDNA 2310042G06 gene 2310042G06Rik 98451_at yes no yes DnaJ (Hsp40) homolog, subfamily B, member 10 Dnajb10 98862_at yes no yes wingless related MMTV integration site 10a Wnt10a 99532_at yes no yes transducer of ErbB-2.1 Tob1 102779_at no yes yes growth arrest and DNA-damage-inducible 45 beta Gadd45b 103443_at no yes yes absent in melanoma 1 Aim1 97906_at no yes yes seven in absentia 2 Siah2 98355_at no yes yes RIKEN cDNA E230012M21 gene E230012M21Rik 100050_at no yes no inhibitor of DNA binding 1 Idb1 100323_at no yes no S-adenosylmethionine decarboxylase 1 Amd1 100979_at no yes no ring finger protein 138 Rnf138 101489_at no yes no S-adenosylmethionine decarboxylase 1 Amd1 101561_at no yes no metallothionein 2 Mt2 101588_at no yes no solute carrier family 16 (monocarboxylic acid transporters), member 1 Slc16a1 102397_at no yes no core-binding factor, runt domain, alpha subunit 2; translocated to, 3 homolog (human) Cbfa2t3h

4 102709_at no yes no histone cell cycle regulation defective homolog A (S. cerevisiae) Hira 102789_at no yes no GATA binding protein 2 Gata2 103375_at no yes no RIKEN cDNA 2810406K24 gene 2810406K24Rik 103748_at no yes no RIKEN cDNA 4933407C03 gene 4933407C03Rik 104186_at no yes no male-specific lethal-2 homolog (Drosophila) Msl2 104492_at no yes no early B-cell factor 3 Ebf3 92614_at no yes no inhibitor of DNA binding 3 Idb3 92672_at no yes no RIKEN cDNA 5530400H20 gene 5530400H20Rik 92722_f_at no yes no sine oculis-related homeobox 1 homolog (Drosophila) Six1 93013_at no yes no inhibitor of DNA binding 2 Idb2 93019_at no yes no H2A histone family, member X H2afx 93130_at no yes no RIKEN cDNA 2600005C20 gene 2600005C20Rik 93134_at no yes no neuronal pentraxin 1 Nptx1 93376_at no yes no Bmp2-inducible kinase Bmp2k 93950_at no yes no forkhead box A2 Foxa2 94483_at no yes no casein kinase II, alpha 2, polypeptide Csnk2a2 94773_at no yes no kallikrein 6 Klk6 94869_at no yes no AE binding protein 2 Aebp2 95360_at no yes no zinc finger, CCHC domain containing 6 Zcchc6 95606_at no yes no synaptotagmin binding, cytoplasmic RNA interacting protein Syncrip 95892_at no yes no ------Adult male aorta and vein cDNA, RIKEN full-length enriched library, clone:A530061A19 product:unknown EST, full 95995_at no yes no --- insert sequence 96489_at no yes no ------96598_at no yes no RIKEN cDNA D430039C20 gene D430039C20Rik 96759_r_at no yes no SEC14-like 2 (S. cerevisiae) Sec14l2 97548_at no yes no PRP39 pre-mRNA processing factor 39 homolog (yeast) Prpf39

5 97970_at no yes no antigen p97 (melanoma associated) identified by monoclonal antibodies 133.2 and 96.5 Mfi2 98350_at no yes no somatostatin receptor 2 Sstr2 98351_g_at no yes no somatostatin receptor 2 Sstr2 98352_r_at no yes no somatostatin receptor 2 Sstr2 98942_r_at no yes no RIKEN cDNA 2310032D16 gene 2310032D16Rik 103891_i_at no no yes elongation factor RNA polymerase II 2 Ell2 104155_f_at no no yes activating transcription factor 3 Atf3 104156_r_at no no yes activating transcription factor 3 Atf3 93320_at no no yes carnitine palmitoyltransferase 1, liver Cpt1a 100346_at yes no no fibroblast growth factor 9 Fgf9 102021_at yes no no interleukin 4 receptor, alpha Il4ra 102373_at yes no no glutamyl aminopeptidase Enpep 102573_at yes no no neurexophilin 1 Nxph1 102798_at yes no no adrenomedullin Adm 103369_at yes no no RIKEN cDNA 9430029L20 gene 9430029L20Rik 103432_at yes no no interferon-stimulated protein Isg20 103599_at yes no no transformed mouse 3T3 cell double minute 1 Mdm1 103806_at yes no no low density lipoprotein-related protein 5 Lrp5 103909_at yes no no copper chaperone for superoxide dismutase Ccs 104041_at yes no no RIKEN cDNA 1810009A16 gene 1810009A16Rik 92247_at yes no no rho GTPase activating protein 5 Arhgap5 92345_g_at yes no no SWI/SNF related, matrix associated, actin dependent regulator of chromatin, subfamily a, member 3 Smarca3 92533_at yes no no calcitonin/calcitonin-related polypeptide, alpha Calca 92707_at yes no no plasmacytoma variant translocation 1 Pvt1 92805_s_at yes no no ADP-ribosylation-like 4 Arl4 92830_s_at yes no no zinc finger protein 36 Zfp36

6 92877_at yes no no transforming growth factor, beta induced Tgfbi 92953_at yes no no formin Fmn 93568_i_at yes no no RIKEN cDNA 2610042L04 gene 2610042L04Rik 93895_s_at yes no no inositol 1,4,5-triphosphate receptor 1 Itpr1 93942_at yes no no inositol polyphosphate-1-phosphatase Inpp1 94037_at yes no no elastase 2 Ela2 94123_at yes no no neurogenic differentiation 4 Neurod4 94171_at yes no no integrin beta 2-like Itgb2l 94642_at yes no no guanosine diphosphate (GDP) dissociation inhibitor 2 Gdi2 94643_at yes no no DNA segment, Chr 7, ERATO Doi 458, expressed D7Ertd458e 95325_at yes no no pleckstrin homology, Sec7 and coiled/coil domains 1 Pscd1 95543_at yes no no tropomyosin 4 Tpm4 95632_f_at yes no no mevalonate kinase Mvk 96679_at yes no no DnaJ (Hsp40) homolog, subfamily B, member 9 Dnajb9 96856_at yes no no complement component 1, q subcomponent binding protein C1qbp 97001_r_at yes no no olfactory receptor 156 Olfr156 97146_g_at yes no no similar to Caenorhabditis elegans protein C42C1.9 Keo4 97172_s_at yes no no ATP-binding cassette, sub-family C (CFTR/MRP), member 9 Abcc9 97264_r_at yes no no casein kinase 1, delta Csnk1d 97317_at yes no no ectonucleotide pyrophosphatase/phosphodiesterase 2 Enpp2 97367_at yes no no A kinase (PRKA) anchor protein 1 Akap1 97369_g_at yes no no A kinase (PRKA) anchor protein 1 Akap1 97964_at yes no no FK506 binding protein 11 Fkbp11 98336_s_at yes no no replication factor C 1 Recc1 98486_at yes no no stathmin-like 4 Stmn4 98552_at yes no no polyamine-modulated factor 1 Pmf1

7 98731_at yes no no RAB5B, member RAS oncogene family Rab5b 98860_at yes no no steroid sulfatase Sts 98952_at yes no no DNA segment, Chr 8, ERATO Doi 325, expressed D8Ertd325e 99414_at yes no no rhophilin, Rho GTPase binding protein 1 Rhpn1 99419_g_at yes no no BCL2-like 11 (apoptosis facilitator) Bcl2l11 99432_at yes no no cytoplasmic linker 2 Cyln2 99639_at yes no no ubiquitin specific protease 10 Usp10 99798_at yes no no T-cell receptor beta, variable 13 Tcrb-V13 99889_at yes no no somatostatin receptor 5 Sstr5 99907_at yes no no regulator of G-protein signaling 9 Rgs9 99984_at yes no no ELK3, member of ETS oncogene family Elk3

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