tissue-specific transcription factor hnf4α inhibits cell proliferation and induces apoptosis in the...

Biol. Chem., Vol. 388, pp. 91–106, January 2007 • Copyright � by Walter de Gruyter • Berlin • New York. DOI 10.1515/BC.2007.011

2007/259

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Tissue-specific transcription factor HNF4a inhibitscell proliferation and induces apoptosis in the pancreaticINS-1 b-cell line

Silke Erdmann1, Sabine Senkel1, Tanja Arndt1,Belen Lucas3, Jorn Lausen2, Ludger Klein-Hitpass1, Gerhart U. Ryffel1

and Heike Thomas1,*1 Universitatsklinikum Essen, Institut fur Zellbiologie,D-45122 Essen, Germany2 Max-Delbruck-Centrum fur Molekulare Medizin,Zelldifferenzierung und Tumorigenese, D-13092 Berlin,Germany3 Schering AG, D-13353 Berlin, Germany

* Corresponding authore-mail: [email protected]

Abstract

Hepatocyte nuclear factor 4a (HNF4a) is a tissue-specifictranscription factor expressed in many cell types, includ-ing pancreatic b-cells. Mutations in the HNF4a gene inhumans give rise to maturity-onset diabetes of the young(MODY1) characterized by defective insulin secretion byb-cells. To elucidate the mechanism underlying this dis-ease, we introduced the splice form HNF4a2 or HNF4a8into the rat b-cell line INS-1. Upon tetracycline-inducedexpression, both HNF4a isoforms caused distinct chang-es in cell morphology and a massive loss of cell numbersthat was correlated with reduced proliferation andinduced apoptosis. This differential activity was reflectedin oligonucleotide microarray analysis that identified moregenes affected by HNF4a2 compared to HNF4a8, andsuggests that both isoforms regulate largely the same setof genes, with HNF4a2 being a stronger transactivator.We verified the induction of selected transcripts by real-time RT-PCR, including KAI1 and AIF, both known tohave apoptotic potential. By establishing cell lines withinducible expression of these target genes, we deducethat both factors are insufficient to induce apoptosis. Wepropose that the anti-proliferative and apoptotic proper-ties of HNF4a may be an essential feature impaired inMODY1 and possibly also in type 2 diabetes.

Keywords: cell multiplication; endocrine pancreas;gene expression; MODY1; tetracycline induction.

Introduction

Hepatocyte nuclear factor 4a (HNF4a), a member of thenuclear receptor superfamily, has a modular structurewith several functional domains, comprising an N-termi-nal activation domain (AF1), a zinc-finger DNA-bindingregion, a ligand-binding and dimerization domain with asecond activation function (AF2), and a C-terminal

repression domain (Sladek and Seidel, 2001). The naturalligands of HNF4a are endogenous fatty acids whichseem to act rather as structural co-factors than as clas-sical regulatory ligands (Dhe-Paganon et al., 2002; Benoitet al., 2004). HNF4a is a cell-specific transcription factorwith expression in liver, kidney and intestine, and loweractivity in pancreas and stomach (Sladek and Seidel,2001; Tanaka et al., 2006). It plays an important role inearly vertebrate development, as homozygous knockoutmice die during early gastrulation due to dysfunction ofthe visceral endoderm (Chen et al., 1994). It is crucial forestablishing and maintaining hepatocyte phenotype (Liet al., 2000; Hayhurst et al., 2001) and liver architecture(Parviz et al., 2003). In hepatocytes, HNF4a regulates theexpression of various genes with functions in nutrienttransport and metabolism, blood coagulation, immuneresponse, xenobiotic detoxification and ureagenesis, aswell as transcription factors, such as HNF1a (Sladek andSeidel, 2001). In the insulin-secreting b-cells of the pan-creas, HNF4a regulates the expression of genes involvedin glucose transport and metabolism, as well as metab-olism-secretion coupling (Stoffel and Duncan, 1997;Wang et al., 2000; Gupta et al., 2005). Heterozygousmutations in the human HNF4a gene give rise to matur-ity-onset diabetes of the young subtype 1 (MODY1), anautosomal dominant inherited form of diabetes mellituscharacterized by early onset, usually before 25 years ofage, and impaired glucose-stimulated insulin secretiondue to pancreatic b-cell dysfunction (Hattersley, 1998;Ryffel, 2001; Fajans et al., 2001). Of the six subtypes ofMODY known so far, five are caused by mutations ingenes encoding transcription factors. Apart from HNF4a

(MODY1) these are HNF1a/TCF1 (MODY3), PDX/IPF1(MODY4), HNF1b/TCF2 (MODY5) and NEUROD1/BETA2(MODY6) (Servitja and Ferrer, 2004). Mutations in thegenes encoding these transcription factors cause pro-gressive b-cell failure in individuals who are born healthyand develop overt hyperglycemia in early adulthood (Hat-tersley, 1998). The transcription factors associated withMODY are connected in a complex cross-regulatory net-work that determines many important b-cell functions(Servitja and Ferrer, 2004). There is evidence of a haploin-sufficiency mechanism of MODY1, as many studies onHNF4a exclude a dominant-negative effect of the mutat-ed factor, but rather imply a loss-of-function mechanism(Stoffel and Duncan, 1997; Sladek et al., 1998; Lausenet al., 2000). Consistent with this assumption, MODY1mutants have been detected in the promoter areas of theHNF4a gene (Thomas et al., 2001; Hansen et al., 2002).In addition, several genetic studies support the assump-tion that HNF4a is also a susceptibility gene for commontype 2 diabetes (reviewed by Gupta and Kaestner, 2004).In fact, gene expression profiling of type 2 diabetes

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92 S. Erdmann et al.


patients compared to normal glucose-tolerant controlsrevealed a substantial decrease in HNF4a mRNA in pan-creatic b-cells of type 2 diabetes patients (Gunton et al.,2005).

Several HNF4a isoforms are known (Sladek and Sei-del, 2001) that are generated by alternative splicing anddifferential promoter usage (Thomas et al., 2001). Splicevariants derived from the P1 and P2 promoters arereferred to as HNF4a1–HNF4a6 and HNF4a7–HNF4a9,respectively. The differential promoters give rise to pro-teins with distinct N-termini. The activation function AF1,contained exclusively in proteins derived from the P1promoter, plays a role in HNF4a transcriptional potentialby recruitment of coactivators and by synergism with theactivation function AF2 (Green et al., 1998; Eeckhoute etal., 2001; Kistanova et al., 2001). The lack of AF1 in P2-derived isoforms of HNF4a is likely the reason for theirdecreased transcriptional activity (Torres-Padilla et al.,2002; Eeckhoute et al., 2003). While the expression ofHNF4a1/2 and HNF4a7/8 in liver has been shown to bedevelopmentally regulated (Torres-Padilla et al., 2001),the relative abundance of the P1- versus P2-derivedsplice forms in pancreatic b-cells is disputed (Boj et al.,2001; Thomas et al., 2001; Hansen et al., 2002; Eeck-houte et al., 2003). HNF4a has been reported to regulateseveral genes in pancreatic b-cells, dysfunction of whichcould, at least partially, explain the impaired glucose-stimulated insulin secretion in diabetic patients. Theseinclude the genes involved in metabolism-secretioncoupling, such as mitochondrial OGDH E1 subunit, UCP-2 (Wang et al., 2000) and the potassium channel subunitKir6.2, as well as the insulin gene (Bartoov-Shifman etal., 2002). Although the dysfunction of all these HNF4a

target genes may be most relevant, none of themexplains the progressive nature of MODY. However,recently it has been reported that HNF4a inhibits cell pro-liferation in various cell types (Chiba et al., 2003; Laza-revich et al., 2004; Lucas et al., 2005).

Therefore, we explored the possibility that HNF4a

exerts this anti-proliferative effect in pancreatic b-cells aswell and thus conditionally overexpressed HNF4a in ratINS-1 cells. As P1- and P2-promoter derived HNF4a iso-forms may both play a role, we used the isoformsHNF4a2 and HNF4a8 in our study.

Results

Establishment of cell lines conditionally expressingHNF4a variants

To investigate the function of transcription factors in pan-creatic b-cells, we recently adapted the rat insulinomacell line INS-1 (Thomas et al., 2004) for site-specific inte-gration of a gene of interest by insertion of a single FRTsite that can be used for Flp recombinase integration.Thus, a single-copy transgene will always be integratedat the same chromosomal FRT site, abolishing the needto select individual cell clones. Furthermore, we integrat-ed the tetracycline repressor, allowing tetracycline induc-tion of the transgene (Thomas et al., 2004). In this studywe used the Flp-In INS-1 cell clone 1-1.2, which has alow HNF4a level and has the most prominent b-cell phe-

notype, as it displays high expression of insulin 1 and 2and a relatively low level of glucagon compared to theparental INS-1 cell line (Thomas et al., 2004). We intro-duced the two HNF4a isoforms, HNF4a2 and HNF4a8,which differ at the N-terminus (Nakhei et al., 1998), aswell as the MODY mutant R154X (Lindner et al., 1997)that displays impaired transcriptional activity (Lausen etal., 2000; Laine et al., 2000) and the artificial dominant-negative mutant C106R (Taylor et al., 1996). All con-structs (Figure 1A) contained an N-terminal myc-tag forimmunochemical detection of the transgene. Two inde-pendent cell lines (referred to as �1 and �2), represent-ing pools of cell clones, were established for eachconstruct and additionally for green fluorescent protein(GFP) as a control. Immunofluorescence showed thatmore than 90% of the cells express HNF4a after 24 h ofinduction with 1 mg/ml tetracycline (Figure 1B). Westernblot analysis demonstrated induction of similar amountsof protein for all HNF4a-expressing cell lines (Figure 1C),as well as a tetracycline dose-dependent induction, asexemplified for HNF4a2�1 (Figure 1D). As maximum pro-tein expression was observed at a concentration of50 ng/ml tetracycline, we used this concentration in allsubsequent experiments.

Expression of HNF4a causes morphologicalchanges and a massive reduction in the numberof INS-1 cells

At 2 days after tetracycline induction of HNF4a2, exten-sive morphological changes in the cells could be obser-ved (Figure 2). They become large, flat and polygonal.Pronounced vacuolization occurred, with many shrunkenand dead cells. In contrast, induction of the isoformHNF4a8 led to completely different morphologicalchanges. After 2 days, the cells started to lose cell-cellcontact, were singled out and grew as isolated cells.Dead cells were visible, but much fewer than for HNF4a2expression. No morphological changes could be ob-served upon induction of the MODY mutant R154X (Fig-ure 2) or the artificial mutant C106R (data not shown).

To measure cell growth, we determined the metabolicactivity using the MTS assay at 1–6 days after tetracy-cline treatment (Figure 3A). The two independent celllines expressing HNF4a2 showed a rapid decrease in cellnumber. After 3 days, the number of induced cells wasapproximately 10% of the number of correspondinguninduced cells and after 4 days there were hardly anycells left. Induction of HNF4a8 also caused a relativedecrease in cell number, even though the effect was notas strong as with HNF4a2. In contrast, expression of themutants C106R (see below) and R154X, as well as GFP,did not affect the cell numbers.

As an apparent transient increase in cell number wasnoted for HNF4a8 after 1 day of induction, we countedthe cells at different time points. As shown in Figure 3B,using this direct measurement, HNF4a8 exclusivelyexhibited a significant decrease in cell number, with areduction to approximately 40% after 4 days. As the MTSassay monitors the metabolic activity of cells, inductionof HNF4a8 probably led to an initial increase in metabolicactivity in the INS-1 cells.



HNF4a inhibits cell proliferation and induces apoptosis 93


Figure 1 Establishment of cell lines conditionally expressingHNF4a derivatives.(A) Schematic drawing of the proteins for which stable inducibleINS-1 cell lines were created using the Flp-In T-REx system. Theprotein domains and the activation functions AF1 and AF2 areindicated (Sladek and Seidel, 2001). All HNF4a variants have amyc-tag at the N-terminus. (B) Immunofluorescence of stableINS-1 Flp-In T-REx cell lines after induction of transgene expres-sion for 24 h with 1 mg/ml tetracycline. The phase contrastimage is shown to the left of each corresponding fluorescenceimage stain of the myc-tagged HNF4a variants. (C) Westernblotting of whole cell extracts of the INS-1 Flp-In T-REx cell lineswith a-myc antibody 9E10. Cells were cultured without (-) or with(q) 1 mg/ml tetracycline for 24 h. As previously observed inHEK293 cells (Lucas et al., 2005) the MODY1 mutant R154X wasexpressed at a much lower level. (D) Tetracycline concentration-dependent transgene induction exemplified for the cell lineHNF4a2�1. Cells were cultured without (-tet) or with the indi-cated concentrations of tetracycline for 24 h and whole cellextracts were then analyzed by Western blotting using the 9E10a-myc antibody.

Figure 2 Morphological changes in INS-1 cells upon HNF4a

overexpression.Phase contrast images of cell lines HNF4a2�2, HNF4a8�1 andR154X�2 cultured without (-) or with (q) tetracycline for the timeindicated.

Expression of HNF4a leads to a decreased rateof INS-1 cell proliferation

To identify the cause of the decreased cell growth uponHNF4a2 and HNF4a8 expression, we measured the cellproliferation rate by incorporation of BrdU for 1 h andsubsequent flow cytometry analysis. As shown in Figure4A, the percentage of cells in S-phase decreased fromapproximately 40% in uninduced cells to 20% after2 days of HNF4a2 expression, implying a reduction incell proliferation caused by HNF4a2. In contrast, nochange in proliferation rate could be observed uponR154X or GFP expression. HNF4a8 expression had adelayed response compared to HNF4a2 expression, asno significant decrease in cells undergoing S-phase after2 days of induction was observed (data not shown) anda decrease was only observed on day 4 of induction (Fig-ure 4B).

Expression of HNF4a induces apoptosisin INS-1 cells

At 3 days after induction of HNF4a2 there were fewercells present than were plated out initially and dying cellswere clearly visible, so we searched for apoptosis usingtwo different approaches.

First cell cycle analysis by flow cytometry was per-formed using propidium iodide staining of DNA. Figure5A shows the cell cycle distribution, with cells in thesubG1-phase corresponding to potentially apoptotic





Figure 3 Reduction in relative cell numbers upon HNF4a

expression.Relative numbers of cells as assessed by MTS assay (A) or cellcounting with a hematocytometer (B) after induction (50 ng/mltetracycline) of transgene expression for different time periods.Cell numbers are given as a percentage of the appropriate unin-duced controls. Data are mean"SD of triplicate determinations. Figure 4 Reduction in cell proliferation rate upon HNF4a

expression.The percentage of cells that have incorporated BrdU and thusentered S-phase after 1 h of incubation is presented. Cells werenot induced (-) or induced (q) with 50 ng/ml tetracycline for 2(A) or 4 (B) days. BrdU incorporation was assessed by flow cyto-metry. Data are mean"SD of triplicates.

cells. The data showed an increase in cells in subG1-phase 2 days after induction of HNF4a2 expression from3% (uninduced) to 17% (induced), while R154X expres-sion had no effect. Cells expressing HNF4a8 failed toexhibit any significant change in cell cycle distributionafter 2 or 4 days of tetracycline treatment (data notshown).

We then measured the activity of the executioner cas-pases 3 and 7, which play a central role in apoptosis. Asshown in Figure 5B, expression of HNF4a2 resulted in asignificant increase in caspase activity as early as1.5 days after induction, and after 3 days a three-foldincrease was measured. Taking into account that there isan approximately ten-fold loss of cell numbers uponHNF4a2 expression after 3 days of induction (Figure 3A),we estimated an approximately 30-fold increase in cas-pase activity per cell. For HNF4a8 overexpression, wecalculated an approximately 2.4-fold increase in caspaseactivity after 2 days of tetracycline treatment, consideringa cell number of ;65% for induced compared to unin-duced cells. The specificity of the effect mediated byHNF4a2 and HNF4a8 proteins was supported by thefinding that neither the MODY mutant R154X (Figure 5B)nor the artificial mutant C106R (see below) led to anincrease in caspase activity.

In conclusion, our two independent assays show thatexpression of HNF4a2 induced apoptosis in INS-1 cells.This result was additionally confirmed by the fact thatexpression of HNF4a2 gives rise to fragmentation of thegenomic DNA into multimers of ca. 200 bp in length,beginning 1.5 days after induction (data not shown). Incontrast, apoptosis induced by expression of the isoformHNF4a8 could only be detected in the caspase 3/7assay, which is the more sensitive assay of the methodsused.

Target genes of HNF4a2 and HNF4a8

To identify candidate genes that mediate the anti-prolif-erative and apoptotic effects of HNF4a, we performedoligonucleotide microarray analysis using the AffymetrixGeneChip RAE230A containing more than 15 000 probesets. To identify potential target genes of HNF4a2 andHNF4a8, we compared the expression profiles of cellstreated with 50 ng/ml tetracycline for 24 h and the cor-responding uninduced cells in the two independent celllines. We considered transcripts as potential targets ifthey showed an at least two-fold increase or decrease inexpression levels in induced compared to uninducedcells in both of the two independent cell lines establishedfor each transcription factor. In addition, candidates hadto score at least one present call in the uninduced versusinduced sample pair. Using these filter criteria, we found257 and 100 transcripts differentially regulated byHNF4a2 and HNF4a8, respectively. For the two R154Xcell lines, only one probe set, encoding the lipid trans-porter ApoC1, passed the filter and this was absent inthe potential target lists for HNF4a2 and HNF4a8. Thisindicates that all probe sets affected by HNF4a2 andHNF4a8 are potential candidates for mediating the anti-proliferative and apoptotic effects. The Venn diagrams inFigure 6 show that the majority of transcripts changedupon HNF4a2 or HNF4a8 induction were upregulated.HNF4a8 affected a markedly smaller number of probesets, with a 70% overlap with HNF4a2-regulated targets,comprising no regulation in the opposite direction. Theentire list of probe sets regulated by HNF4a2 and





Figure 5 Induction of apoptosis upon expression of HNF4a.(A) Cell cycle distribution of Flp-In T-REx INS-1 cells HNF4a2�1 (left panel) and R154X�2 (right panel) without (-tet) or with additionof 50 ng/ml tetracycline (qtet) for 2 days. Cells were harvested, fixed and DNA content analysis was performed by flow cytometryafter propidium iodide staining. The percentage of cells in S/G2-phase and in subG1-phase (indicating apoptotic cells) is presented.(B) The indicated Flp-In T-REx INS-1 cell lines were treated with 50 ng/ml tetracycline for the time indicated, lysed and caspase 3/7activity was assessed. The fold induction was calculated using appropriate uninduced cells as a reference. Data are mean"SD oftriplicates.

Figure 6 Transcripts affected by HNF4a2 and HNF4a8 asassessed by microarray analysis.Venn diagrams representing the probe sets for which expressionwas regulated by HNF4a2 and HNF4a8 after 24-h induction with50 ng/ml tetracycline. Numbers in the overlapping segmentsrepresent common probe sets and numbers in parentheses indi-cate the total number of probe sets affected by each transcrip-tion factor.

HNF4a8 is included in the Appendix (Tables A1–A6). Thedata are arranged according to the Venn diagram in Fig-ure 6 and the fold-induction refers to the average for thetwo independent cell lines.

Approximately 50% of the changed probe sets areannotated to specific genes according to Affymetrixrecords (http://www.affymetrix.com/analysis/index.affx).

Assigning the potential target genes to the categoriesdefined by their gene ontology annotations (Ashburner etal., 2000), ‘metabolism’ was mainly affected by HNF4a2,as previously observed (Thomas et al., 2004). ForHNF4a8, only 46 of the differentially regulated probe setswere annotated to specific genes, which was too low fora corresponding statistical analysis.

Since HNF4a2 showed a significantly stronger effecton cell proliferation and apoptosis, we focused on probesets regulated by this isoform to search for candidategenes mediating these effects. According to the geneontology annotations, we choose two probe sets regu-lated by HNF4a2 that were assigned to cell proliferation,namely cyclin D1 (Ccnd1) and apoptosis-inducing factor1 (AIF/Pdcd8). Among the probe sets allocated to pro-grammed cell death, we choose nerve growth factorreceptor (ngfr) and again AIF. However, we excludedcyclin D1 for further analysis, as the corresponding probeset sequence scored no exact match in BLAST andcyclin D1 is represented by two additional probe sets thatare not significantly changed by HNF4a2 or HNF4a8expression. Since the majority of transcripts have no GOannotations, we additionally searched the data for upre-gulated probe sets connected to apoptosis and down-regulated probe sets connected to cell proliferation usingOMIM and PubMed. We thus identified KAI1 (kangai),which has been shown to induce apoptosis in differentcell lines (Schoenfeld et al., 2003). We selected the threetranscripts (AIF, ngfr and KAI1) that are candidates for





Figure 7 Real-time RT-PCR verification of microarray candi-date genes.The abundance of seven transcripts indicated by microarrayanalysis to be regulated by HNF4a2, HNF4a8 or both, in Flp-InT-REx INS-1 cell lines, was assessed by real-time RT-PCR. TotalRNA was isolated from cells 24 h after tetracycline (50 ng/ml)treatment and the fold induction was calculated using the appro-priate uninduced cells as a reference. The diagrams show themicroarray data and the real-time RT-PCR data for each cell lineanalyzed.

mediating the anti-proliferative and apoptotic effects forverification by real-time RT-PCR of regulation by HNF4a.We also included in this verification four transcripts withdifferent biological functions, one of which was affectedby both isoforms (asialoglycoprotein receptor 1) andsome that were only or predominantly affected byHNF4a2 (neuromedin B receptor) or HNF4a8 walanyl(membrane) aminopeptidase and transthyretinx.

As shown in Figure 7, six out of seven transcriptsinvestigated by real-time RT-PCR were confirmed to be

regulated. Only ngfr could not be verified, since inHNF4a8�1 cells it showed slight up- instead of down-regulation, as observed in microarray analysis, and, evenmore importantly, it was downregulated in both R154Xcell lines (Figure 7A). These findings disprove the hypoth-esis that ngfr mediates the apoptotic effects. Concerningthe other two candidates involved in cell multiplication,i.e., KAI1 and AIF, real-time RT-PCR confirmed themicroarray data. KAI1 transcripts were increased byHNF4a2 and HNF4a8, but not by the R154X mutant (Fig-ure 7C). In contrast, AIF transcripts were exclusivelyincreased by HNF4a2 and not by the isoform HNF4a8 orthe mutant R154X (Figure 7B). This confirmed theHNF4a2 isoform-specific induction of AIF. For the asia-loglycoprotein receptor (asgr, Figure 7D), neuromedin Breceptor (Figure 7E), alanyl (m) aminopeptidase (Figure7F) and transthyretin (Figure 7G), real time RT-PCR anal-ysis confirmed regulation of the transcripts by HNF4a.However, exclusive or predominant regulation by one ofthe isoforms could not be verified. The extensive differ-ences in fold inductions observed are mostly caused byvery small values of the uninduced probes, resulting inextensive variation of the fold induction.

Candidate target genes for mediating the apoptoticeffects of HNF4a

To explore whether the activation of KAI1 and AIF couldmediate HNF4a-induced apoptosis, we created twoindependent cell lines conditionally overexpressing eachof these proteins using the Flp-In cell line 1-1.2 asdescribed above. The constructs used, human KAI1 andmouse AIF shown in Figure 8A, were inserted into theappropriate vector to allow Flp recombinase-mediatedintegration into the FRT site. Figure 8B demonstrates thatthe KAI1 transcripts were five- to six-fold induced upontetracycline treatment in the two cell lines created(KAI1�1 and KAI1�2). Figure 8C shows tetracyclineinduction of the AIF protein in Western blot analysis. Theinduced protein corresponding to the mouse AIF-EGFPfusion protein of approximately 100 kDa could easily bedistinguished from endogenous AIF protein of 61 kDa.The weaker signal of the upper band may be explainedby the fact that the a-AIF polyclonal antibody targets theC-terminus of AIF, which is linked to EGFP in the fusionprotein.

Morphological inspection of INS-1 cells expressingKAI1 revealed round cells that start to grow in foci (datanot shown). This change in morphology was accompa-nied by a decrease in cell number compared to unin-duced cells after 4 days, as demonstrated by MTSmeasurement (Figure 8D). However, no further decreasein relative cell number could be observed after 5 or6 days. A distinct change in morphology was observedupon induction of AIF. INS-1 cells became slightly smallerand bipolar (data not shown). Under these conditions, aprogressive decrease in relative cell number could bedetected, as shown in Figure 8D, but to a much lesserextent than for HNF4a expression. Significantly, no deadcells could be observed upon induction of KAI1 or AIF.In agreement with this lack of apoptotic cells, no induc-tion of caspase 3/7 activity could be determined for two





Figure 8 Conditional expression of AIF and KAI1.(A) Schematic presentation of the hKAI1-HA protein and mAIF-EGFP fusion protein of human and murine origin, respectively, forwhich stable inducible Flp-In T-REx cell lines were established. tm, transmembrane region; HA, hemagglutinin; MLS, mitochondriallocalization signal; NLS, nuclear localization signal; EGFP, enhanced green fluorescent protein. (B) Induction of hKAI1 expressionassessed by real-time RT-PCR 24 h after addition of 50 ng/ml tetracycline. The Taq-Man assay used is specific for the human protein.Fold inductions were calculated using the corresponding uninduced cells as a reference. (C) Western blot analysis of whole extractsof Flp-In T-REx cells containing mAIF-EGFP as the gene of interest. Cells were not treated (-) or treated (q) with 50 ng/ml tetracyclinefor 24 h and probed with an anti-AIF antibody (Santa Cruz). H1luc�1 is a Flp-In T-REx INS-1 cell line that does not contain mAIF asa gene of interest and was used as a control. (D) Relative numbers of viable cells detected by MTS assay. Cells of the Flp-In T-RExINS-1 cell lines indicated were treated with 50 ng/ml tetracycline to induce expression of the corresponding transgene. Cell numbersare given as a percentage of the appropriate uninduced controls. Data are the mean"SD of triplicate determinations. Data for thecell lines HNF4a2�1 and HNF4a2�2 were taken from a different experiment (Figure 3A) and added for comparison. (E) The Flp-InT-REx INS-1 cell lines indicated were treated with 50 ng/ml tetracycline, lysed and caspase 3/7 activity was assessed. The foldinduction was calculated using the appropriate uninduced cells as a reference. The data are mean"SD of triplicates.

independent AIF or KAI1 cell lines upon tetracyclinetreatment (Figure 8E).

In conclusion, overexpression of neither KAI1 nor AIFhad effects on cell morphology or cell growth compara-ble to HNF4a, and induction of apoptosis could not bedetected.

Discussion

We used the Flp-In T-REx INS-1 host cell line (Thomaset al., 2004) to induce HNF4a2 and HNF4a8 expressionin a reproducible way (Figure 1). Induced expression ofboth isoforms caused distinct morphological changes in





INS-1 cells (Figure 2) and a clear decrease in cell num-bers, which was stronger for HNF4a2 than for HNF4a8(Figure 3). Consistent with the appearance of dead cellsafter HNF4a2 induction, we could confirm that HNF4a2induced apoptosis by cell cycle analysis and measure-ment of caspase 3/7 activity. A smaller increase in cas-pase 3/7 activity, indicating apoptosis, could also bedetected for HNF4a8, but cell cycle analysis did not con-firm these results. As measurement of caspase activity isfar more sensitive than cell cycle analysis, this mightreflect the lower response evoked by this isoform. BrdUincorporation indicated a decreased proliferation rateupon induction of HNF4a2 or HNF4a8 expression, withthe latter showing a delayed response. The anti-prolifer-ative and apoptotic effects of HNF4a were specific, asboth HNF4a mutants and GFP had no effect. The rele-vance of these findings for the development of MODY isimplied by the lack of response to the naturally occurringMODY1 mutant R154X, a truncated protein lacking mostof the ligand-binding and dimerization domain (Figure1A). The artificial mutant C106R, which contains a mis-sense mutation in the zinc-finger motif that abolishesDNA binding (Taylor et al., 1996), also had no effect, thusruling out the possibility that lack of a protein domainexplains the missing effects of R154X. Therefore, we canexclude a posttranscriptional mechanism such as directinhibition of Cdks by protein-protein interaction, asfound, for example, for the transcription factor C/EBPa

in regulating cell proliferation (Wang et al., 2001). Werather conclude that the transcriptional activity of HNF4a

is needed to inhibit cell proliferation and to induce apop-tosis in INS-1 cells.

Microarray analysis revealed 257 and 100 probe setsregulated by HNF4a2 and HNF4a8, respectively. Theresults for HNF4a2 differ from our previous analysis ofthe same cell lines, whereby a greater number of targetgenes was identified, even though more stringent criteriawere used (Thomas et al., 2004). The difference can beexplained by the higher concentration of tetracycline(1 mg/ml) used for induction. However, since cell prolif-eration and apoptosis are regulated by HNF4a expres-sion induced with 50 ng/ml tetracycline, the alteredtranscripts should include the relevant genes. The factthat there are more than twice as many probe sets affect-ed by HNF4a2 than by HNF4a8 agrees with studiesreporting that HNF4a2 is a stronger transactivator, mostlikely due to the presence of the activation function AF1(Nakhei et al., 1998; Torres-Padilla et al., 2002; Eeck-houte et al., 2003; Ihara et al., 2005). Although approxi-mately one-third of the probe sets affected by HNF4a8were not regulated by HNF4a2, the more reliable real-time RT-PCR could not confirm the preferential regulationof two probe sets regulated exclusively by HNF4a8according to our microarray data walanyl (m) aminopep-tidase and transthyretin in Figure 7F,Gx. Our data suggestthat HNF4a2 and HNF4a8 largely regulate the same setof genes, with HNF4a2 being a stronger transactivator.

Many target genes of HNF4a are already known, butmost of these are in the liver (Sladek and Seidel, 2001)and in an embryonic kidney cell line (Lucas et al., 2005).In b-cells of the pancreas, target genes of HNF4a havebeen identified in INS-1 cells using a conditional HNF4a

expression system distinct to our approach (Wang et al.,2000). These target genes include Glut-2, L-type pyru-vate kinase, 2-oxoglutarate dehydrogenase E1 subunit,aldolase B and HNF1a. All of these genes are repre-sented on our microarray and were scored as active, butonly aldolase B was regulated by HNF4a (Table A1).Using a higher tetracycline concentration of 1 mg/ml forinduction, Glut-2 expression was also activated (Thomaset al., 2004). The upregulation reported for HNF1a (Wanget al., 2000) is quite small and is therefore not detectableusing our criteria. Recently, gene expression has beenanalyzed in islets of mice carrying a b-cell-specificHNF4a knockout (Gupta et al., 2005). Surprisingly, noneof the known target genes of HNF4a in b-cells (Wang etal., 2000) are differentially expressed, except for L-typepyruvate kinase. The data from knockout mice also implythat in b-cells the KCNJ11 gene encoding the potassiumchannel subunit Kir6.2 is a direct target of HNF4a andthat its impaired expression is responsible for the phe-notype of glucose intolerance and hyperinsulinemia ofthe knockout mice. In contrast, in our cell system, Kir6.2transcripts were downregulated by HNF4a2 expression.This may reflect the lack of a regulatory system in ourcell culture system compared to the mouse model, inwhich the whole organism is considered. However, asRIP-Cre mice may display glucose intolerance per se(Lee et al., 2006), it is possible that some differentiallyregulated genes are a feature of the RIP-Cre mice andnot a direct effect due to HNF4a downregulation in theislets.

Our data revealed only two genes, AIF and KAI1, con-nected to proliferation or apoptosis. AIF is a death effec-tor that resides in the mitochondrial intermembranespace and translocates to the nucleus upon apoptosisinduction (Susin et al., 1999; Ye et al., 2002). Extramito-chondrially targeted AIF and overexpression of AIF havebeen shown to induce apoptosis in COS cells (Loeffleret al., 2001). KAI1 induces cell death in many differentcell types (Schoenfeld et al., 2003) and was reported tobe important for the prevention of tumor progression andmetastases (Wu et al., 2004). However, overexpressionof neither AIF nor KAI1 in INS-1 cells caused effects sim-ilar to those of HNF4a. The reduction in cell growth wasmuch smaller than for HNF4a (Figure 8D), morphologicalchanges were different, with no dead cells visible (datanot shown), and no increase in caspase activity could bedetected for either protein (Figure 8E). As it was recentlyreported that induction of apoptosis by AIF is cell type-dependent and does not involve the activation of cas-pases, but is rather accompanied by inactivation ofcaspases (Modjtahedi et al., 2006), the role of AIF inapoptosis remains open in our experimental system.Nevertheless, our data exclude the possibility that AIFand KAI1 are able to elicit the HNF4a-mediated apoptoticand anti-proliferative effects in INS-1 cells, but it remainspossible that they cooperate with other proteins. Weshowed an inhibitory effect of HNF4a on proliferation ofthe b-cell line INS-1 and this extends recent studies inthe hepatoma cell line fgHCC (Lazarevich et al., 2004),the embryonic carcinoma cell line F9 (Chiba et al., 2005)and the human embryonic kidney cell line HEK293 (Lucaset al., 2005). In addition, we observed an induction of





apoptosis in b-cells, which this adds an important newaspect to the functional properties of HNF4a. Using RNAinterference, we downregulated the endogenous HNF4a

level in INS-1 cells to approximately 50% within 6 days,but failed to induce accelerated proliferation of the cells(data not shown). This could simply reflect the fact thatINS-1 cells have been adapted in culture to the maximalrate of proliferation.

The regulation of b-cell proliferation and survival withinthe organism is still poorly understood, even though it isof great importance for understanding of the pathogen-esis of diabetes. There is a continued turnover of b-cellsthroughout human life, and increased insulin demand, forexample, in obesity, can be compensated for by anincrease in b-cell mass (Dickson and Rhodes, 2004).Recent observations in diabetes type 2 patients haveshown a relative b-cell loss and increased apoptosis ofb-cells (Butler et al., 2003). This would argue thatimpaired cell proliferation and increased apoptosis arecausative events in diabetes, and thus overfunction ofHNF4a would be predicted rather than a loss of function.Although corresponding data for MODY patients are notavailable, we postulate that the observed decrease in b-cells in diabetes patients may be a secondary phenom-enon, caused, for example, by the toxic effect ofhyperglycemia on b-cells (Robertson, 2004). Based onour data indicating that HNF4a induces apoptosis andinhibits proliferation, we speculate that HNF4a-deficientcells escape its apoptotic and anti-proliferative control.This would alter the b-cell population and is consistentwith the marked decrease in HNF4a transcripts recentlyreported in human pancreatic islets isolated from sub-jects with type 2 diabetes (Gunton et al., 2005). There-fore, our hypothesis may trigger novel clinical investi-gations to explore proportional increases with time incells with dysfunctional HNF4a in MODY, as well as intype 2 diabetes patients.

Materials and methods

Plasmid constructs

The vectors pFRT/lacZeo2 and pcDNA6/TR used to establishthe INS-1 Flp-In T-REx host cell lines and the plasmid pcDNA5/FRT/TO were obtained from Invitrogen (Karlsruhe, Germany). Forsite-specific integration of the transcripts encoding HNF4a2,HNF4a8 and R154X, the corresponding ORF of the humancDNA was inserted in the pcDNA5/FRT/TO vector. MycHNF4a2and mycR154X were derived as NotI fragments from the pOP-mycHNF4a and pOP-mycR154X expression vectors (Lausen etal., 2000), respectively, and cloned into the NotI site of pcDNA5/FRT/TO. The P2 promoter-derived 59-end of HNF4a was ampli-fied by PCR from cDNA obtained from HepG2 RNA using theoligonucleotides 59-CGG GGT ACC ATG GTC AGC GTG AACGCG-39 and 59-CCG GCT AAA TCT GCA GGA G-39 as forwardand reverse primers, respectively (restriction sites used in bold).The PCR product was digested with KpnI and XhoI and the frag-ment was used to replace the corresponding P1 promoter-derived KpnI/XhoI fragment in the pOP-mycHNF4a expressionvector (Lausen et al., 2000). The mycHNF4a8 sequence wasinserted as a NotI fragment into the NotI site of the pcDNA5/FRT/TO plasmid. The sequence of the final construct was veri-fied by sequencing. The mutant C106R was generated byperforming site-directed mutagenesis on the pcDNA5/FRT/TO

HNF4a2 construct (Quick Change site-directed mutagenesis kit;Stratagene, La Jolla, USA) using the primer pair 59-CCA GTGCCG CTA CCG CAG GCT CAA G-39 and 59-CTT GAG CCT GCGGTA GCG GCA CTG G-39. The presence of the mutation wasverified by sequencing.

GFP was derived as a HindIII/XhoI fragment from pCSGFP2(Ryffel and Lingott, 2000) and inserted into the HindIII/XhoI siteof the pcDNA5/FRT/TO plasmid.

Human KAI1-HA was derived as a KpnI-NotI fragment from aCD82-HA plasmid (Schoenfeld et al., 2003), a kind gift from Dr.Stefan Grimm, and cloned into the KpnI/NotI-digested pcDNA5/FRT/TO plasmid. Murine AIF1-EGFP was excised with PmeIfrom the pcDNA3.1q construct (Loeffler et al., 2001), a kind giftfrom Prof. Guido Kroemer, and cloned into the PmeI site ofpcDNA5/FRT/TO.

The Flp expression vector used (pCSFLPe) has beendescribed elsewhere (Werdien et al., 2001).

Establishment of INS-1 Flp-In T-REx cell lines

Establishment of the Flp-In T-REx INS-1 host cell line used, 1-1.2, was as previously described (Thomas et al., 2004). INS-1cell line 1-1.2 was cultured at 378C in RPMI 1640 medium sup-plemented with 10% heat inactivated fetal calf serum, penicillin(100 U/ml), streptomycin (100 U/ml), 2 mM glutamine, 1 mM sodi-um pyruvate, 10 mM HEPES, 50 mM mercaptoethanol, Zeocin�

(200 mg/ml) and blasticidin (10 mg/ml). Stable INS-1 Flp-In T-REx cell lines carrying the inducible transgenes were generatedessentially as described in the Flp-In� T-REx� Core Kit Manual(Invitrogen). Co-transfection of the Flp expression vectorpCSFLPe with the pcDNA5/FRT/TO vector containing the geneof interest was carried out using lipofectamine and hygromycin(150 mg/ml) selection.

Western blotting and immunofluorescence

The anti-myc tag antibody 9E10 was used for detection of myc-conjugated proteins, and anti-AIF antibody (sc-9416; SantaCruz, Heidelberg, Germany) was employed for detection of AIF.For Western blots, peroxidase-coupled sheep anti-mouse immu-noglobulin antibody (Amersham Biosciences, Munich, Germany;HRP-linked whole Ab, NXA931) and peroxidase-coupled mouseanti-goat/sheep immunoglobulin antibody (Sigma-Aldrich,Munich, Germany, A9452) were employed as secondary anti-bodies for detection of myc-conjugated proteins and AIF,respectively and detected using the ECL system (AmershamBiosciences). For immunofluorescence, Cy3-conjugated rat anti-mouse wDianova, Hamburg, Germany; F(ab9)2-fragment, �415-166-166x and rabbit anti-goat wDianova; F(ab9)2-fragment,�305-166-045x antibodies were used as secondary antibodiesfor detection of myc and AIF, respectively.

Cell growth analysis

Relative cell numbers were assessed by hematocytometercounting or using the CellTiter 96� AQueous One Solution CellProliferation Assay from Promega (Mannheim, Germany). Cellswere plated at a density of 32 000 cells/cm2 in 96- or 6-wellplates.

Flow cytometry

Flow cytometry measurements were performed using a FACSVantage� Fluorescence Activated Cell Sorter (Becton Dickinson,Heidelberg, Germany). For PI analysis, cells were harvested andfixed in ice-cold 70% ethanol. After washing with PBS, cellswere suspended in 1 ml of PBS containing 10 mg/ml propidiumiodide (Sigma-Aldrich) and 50 mg/ml RNase and submitted to





flow cytometry. BrdU incorporation experiments were performedusing the In Situ Cell Proliferation Kit, FLUOS from Roche-Diag-nostics (Mannheim, Germany), according to the manufacturer’sprotocol. Cells were incubated with BrdU for 1 h and incorpo-ration was measured by flow cytometry.

Caspase activity

Caspase 3/7 activity was measured using the Caspase-Glo�

3/7 Assay from Promega. Cells were plated at a density of32 000 cells/cm2 in white-walled 96-well plates. Prior to meas-urement in a luminometer (GENios multimode research reader;Tecan, Crailsheim, Germany) cells were incubated with Cas-pase-Glo� 3/7 reagent for 1 h.

Oligonucleotide microarray analysis

Microarray analysis was performed using the Affymetrix (HighWycombe, UK) GeneChip platform as previously described(Thomas et al., 2004). Target identification was restricted toprobe sets that received at least one Present detection call inthe uninduced/induced sample pair. Probe sets exhibiting a sig-nal log2 ratio G1 or F1 (corresponding to two-fold up- or down-regulation) were identified by filtering using the Affymetrix DataMining Tool 3.0.

Real-time RT-PCR

Real-time RT-PCR was performed using TaqMan� Gene Expres-sion Assays (Applied Biosystems, Foster City, CA, USA) as

described by the manufacturer employing the GeneAmp 5700Sequence Detection System. The assays used were:Rn00561634_m1 (ngfr), Rn00442540_m1 (AIF), Rn00582491_m1(KAI1), Rn00561634_m1 (asgr1), Rn00562994_m1 (neuromedinB receptor), Rn00578763_m1 walanyl (m) aminopeptidasex,Rn00562124_m1 (KAI1) and Hs00174463_1 (hKAI1). cDNA wasgenerated with random primers using the Omniscript RT Kit(Qiagen, Hilden, Germany) using 1 mg of total RNA in a 20-mlreaction volume.

Acknowledgments

We greatly acknowledge the technical assistance of NadineEsser and Adriane Parchatka for microarray analysis. We thankKlaus Lennartz for assistance with flow cytometry measure-ments and Noel Morgan for critical reading of the manuscript.This work was supported by the Deutsche Forschungsgemeins-chaft (TH 799/1-1).

Appendix

Probe sets are arranged according to the Venn diagrams (Figure6). The probe sets are the reference numbers of the Affymetrixoligonucleotide microarray RAE230A. Fold induction in columns2 and 3 is given as average value of the two cell lines analyzed.Gene symbols are indicated where available.

Table A1 Probe sets upregulated by HNF4a2 and HNF4a8.

Probe set HNF4a2 HNF4a8 Gene symbol Gene name

1387314_at 136.4 5.3 Sult1b1 Sulfotransferase family 1B, member 11370149_at 128.3 23.4 Asgr1 Asialoglycoprotein receptor 11371951_at 45.4 2.7 – –1377048_at 26.4 4.3 RGD1311026 Similar to cDNA sequence BC021917 predicted1367917_at 25.5 8.7 Cyp2d26 Cytochrome P450, family 2, subfamily d, polypeptide 261370547_at 15.2 13.3 Pzp Pregnancy-zone protein1370725_a_at 12.7 13.6 G6pc Glucose-6-phosphatase, catalytic1372170_at 10.6 2.7 Acy1 Aminoacylase 11367647_at 9.7 5.4 Serpina1 Serine (or cysteine) proteinase inhibitor, clade A

(a-1 antiproteinase, antitrypsin), member 11377821_at 9.6 5.2 Erbb3 V-erb-b2 erythroblastic leukemia viral oncogene homolog 3 (avian)1370511_at 9.6 5.1 Fgb Fibrinogen, B b polypeptide1386944_a_at 9.4 8.3 G6pc Glucose-6-phosphatase, catalytic1387022_at 8.6 8.4 Aldh1a1 Aldehyde dehydrogenase family 1, member A11385243_at 8.1 2.4 Maf V-maf musculoaponeurotic fibrosarcoma (avian) oncogene

homolog (c-maf)1368397_at 7.9 3.3 Ugt2b5//Ugt2b4 UDP-glucuronosyltransferase 2 family, member 5//

UDP glycosyltransferase 2 family, polypeptide B41390042_at 7.7 3.5 MGC109491 Similar to 1110007F12Rik protein1389206_at 7.7 4.7 – –1368085_at 7.6 6.1 Gchfr GTP cyclohydrolase I feedback regulator1387375_at 7.1 2.4 Khk Ketohexokinase1387209_at 7.1 2.4 Rgpr Regucalcin gene promotor region related protein1388213_a_at 6.8 3.4 RT1.S3 RT1 class Ib, locus S31370220_at 6.6 3.2 Scpep1 Serine carboxypeptidase 11388102_at 6.2 2.5 Ltb4dh Leukotriene B4 12-hydroxydehydrogenase1373309_at 6.1 5.4 – Similar to RIKEN cDNA 1810054O13 (predicted)1374948_at 5.6 2.4 RGD1309043 Similar to hypothetical protein MGC37887 predicted1372328_at 5.0 2.5 Knsl8 Kinesin-like 8 (predicted)1371974_at 4.9 3.1 Phyhd1 Phytanoyl-CoA dioxygenase domain containing 1 predicted1371913_at 4.9 2.8 Tgfbi Transforming growth factor, b induced1398370_at 4.9 2.9 – –1374452_at 4.9 11.1 Pde9a Phosphodiesterase 9A





Table A1 (Continued)


1388190_at 4.7 2.9 Apob Apolipoprotein B1376226_at 4.6 3.6 – Similar to Ab2-0761367843_at 4.4 3.4 RGD:620311 Aldo-keto reductase family 7, member A2

(aflatoxin aldehyde reductase)1371123_x_at 4.3 2.0 RT1.S3 RT1 class Ib, locus S31370299_at 4.1 4.1 Aldob Aldolase B1389219_at 4.1 2.4 – NIMA (never in mitosis gene a)-related expressed kinase 21389744_at 3.9 2.2 LOC499898 LOC4998981372841_at 3.9 2.4 Dp1l1 Deleted in polyposis 1-like 1 predicted1386976_at 3.8 2.6 Kai1 Kangai 11390454_at 3.8 2.2 Nipsnap1 4-Nitrophenylphosphatase domain and non-neuronal

SNAP25-like protein homolog 1 (C. elegans) predicted1390383_at 3.8 2.8 RGD:1359288 Adipose differentiation-related protein1370365_at 3.8 3.0 Gss Glutathione synthetase1388537_at 3.7 2.2 Nipsnap1 4-Nitrophenylphosphatase domain and non-neuronal

SNAP25-like protein homolog 1 (C. elegans) predicted1388212_a_at 3.7 2.6 RT1.S3 RT1 class Ib, locus S31376191_at 3.5 2.4 Hpgd 15-Hydroxyprostaglandin dehydrogenase1372997_at 3.5 2.3 Csnk2b Casein kinase 2, b subunit1377155_at 3.2 2.4 – Similar to RIKEN cDNA 1700019G171387253_at 3.1 4.9 Guca2b Guanylate cyclase activator 2b1387867_at 3.0 2.3 – –1387859_at 3.0 2.3 Nifs Nitrogen fixation gene 1 (S. cerevisiae)1372681_at 3.0 2.6 – –1372101_at 3.0 3.8 Ppap2b ER transmembrane protein Dri 421387631_at 3.0 2.3 Hpgd 15-Hydroxyprostaglandin dehydrogenase1389354_at 3.0 2.7 LOC362057 Similar to semaF cytoplasmic domain associated protein 21369073_at 2.9 2.5 Nr1h4 Nuclear receptor subfamily 1, group H, member 41388720_at 2.9 2.3 – Hypothetical LOC3612251383906_at 2.8 2.6 RGD:1359633 Similar to lung inducible neuralized-related C3HC4 RING

finger protein1375934_at 2.7 2.1 LOC315911 Similar to RIKEN cDNA D330045A201371030_at 2.6 2.5 Spp2 Secreted phosphoprotein 21367995_at 2.6 2.3 Cat Catalase1388710_at 2.6 2.2 – Ras responsive element binding protein 1 (predicted)1387793_at 2.5 2.3 Slc9a3r1 ERM-binding phosphoprotein1374222_at 2.5 2.7 RGD:1303323 Tumor suppressing subtransferable candidate 51369954_at 2.5 2.2 Idh1 Isocitrate dehydrogenase 1 (NADPq), soluble1367791_at 2.2 2.0 Ramp1 Receptor (calcitonin) activity modifying protein1

Table A2 Probe sets downregulated by HNF4a2 and HNF4a8.


1375861_at 0.30 0.40 – –1373035_at 0.45 0.36 – –

Table A3 Probe sets upregulated by HNF4a2 only.

Probe set HNF4a2 Gene symbol Gene name

1389067_at 20.0 Slco4a1 Solute carrier organic anion transporter family, member 4a11371209_at 19.6 RT1-CE5 RT1 class I, CE51367994_at 18.7 Dpyd Dihydropyrimidine dehydrogenase1367909_at 17.9 Dcxr Dicarbonyl L-xylulose reductase1377135_at 14.5 – –1387239_a_at 13.6 Padi4 Peptidyl arginine deiminase, type IV1371697_at 12.4 RGD1309044 Similar to RIKEN cDNA 0610039C21 (predicted)1369985_at 10.7 Crybb1 Crystallin, b B11368092_at 10.6 Fah Fumarylacetoacetate hydrolase1368915_at 10.2 Kmo Kynurenine 3-monooxygenase (kynurenine 3-hydroxylase)1377223_at 9.7 – –1374648_at 9.3 – –1373307_at 8.8 RGD1308823 Similar to RIKEN cDNA 2810468K17 (predicted)1376615_at 7.9 Tead3 TEA domain family member 3 (predicted)1369373_at 7.4 Fgfr3 Fibroblast growth factor receptor 31387515_at 7.0 Nmbr Neuromedin B receptor1376327_at 6.8 Tnfrsf14 Tumor necrosis factor receptor superfamily, member 14 (herpesvirus entry

mediator) (predicted)







1368358_a_at 6.6 Ptprr Protein tyrosine phosphatase, receptor type, R1373108_at 6.1 Ppp1r3c Protein phosphatase 1, regulatory (inhibitor) subunit 3C (predicted)1387445_at 5.6 Phkg1 Phosphorylase kinase g 11370818_at 5.2 Decr2 2-4-Dienoyl-coenzyme A reductase 2, peroxisomal1368045_at 5.1 RGD:620059 Solute carrier family 31 (copper transporters), member 11380600_at 5.0 – –1372672_at 4.9 Qprt predicted Quinolinate phosphoribosyltransferase (predicted)1368593_at 4.8 Cd1d1 CD1d1 antigen1375954_at 4.8 S100a13 S100 calcium binding protein A13 (predicted)1388534_at 4.6 Slc31a1 Solute carrier family 31 (copper transporters), member 11368622_at 4.6 Fbp2 Fructose-1,6-bisphosphatase 21371171_at 4.6 RT1-Aw2 RT1 class Ib, locus Aw21368046_at 4.5 RGD:620059 Solute carrier family 31 (copper transporters), member 11390399_at 4.5 Crebl2 cAMP responsive element binding protein-like 2 (predicted)1368659_at 4.4 Agxt2 Alanine-glyoxylate aminotransferase 21367673_at 4.0 Selenbp1 Selenium binding protein 21374200_at 3.9 Slc29a3 Solute carrier family 29 (nucleoside transporters), member 31374440_at 3.9 RGD:1303235 Dehydrogenase/reductase (SDR family) member 81373732_at 3.9 Acp6 Acid phosphatase 6, lysophosphatidic (predicted)1376765_at 3.8 LOC361348 Similar to maestro1369110_x_at 3.8 RT1-Aw2 RT1 class Ib, locus Aw21371659_at 3.7 Rhoc ras homolog gene family, member C (predicted)1373512_at 3.7 Ilvbl ilvB (bacterial acetolactate synthase)-like (predicted)1388913_at 3.6 Ppap2c Phosphatidic acid phosphatase type 2c1389358_at 3.6 – NIMA (never in mitosis gene a)-related expressed kinase 21389085_at 3.6 – –1377079_a_at 3.5 Ppox Protoporphyrinogen oxidase (predicted)1369169_at 3.5 Slc23a1 Solute carrier family 23 (nucleobase transporters), member 11387038_at 3.5 Ccs Copper chaperone for superoxide dismutase1388812_at 3.4 LOC287550 Similar to novel protein1372302_at 3.4 RGD1308697 Similar to hypothetical protein FLJ10579 (predicted)1373118_at 3.4 – –1387826_at 3.4 LOC361819 Similar to pyridoxal (pyridoxine, vitamin B6) kinase1376808_at 3.3 – –1374478_at 3.3 RGD1305347 Similar to RIKEN cDNA 2610528J11 (predicted)1369412_a_at 3.3 Slc19a1 Solute carrier family 19, member 11387508_at 3.2 Baat Bile acid-coenzyme A:amino acid N-acyltransferase1368514_at 3.2 Maob Monoamine oxidase B1368467_at 3.2 Cyp4f2 Cytochrome P450, family 4, subfamily F, polypeptide 21367735_at 3.2 Acadl Acetyl-coenzyme A dehydrogenase, long-chain1388587_at 3.2 RGD:1303321 Immediate early response 31388539_at 3.2 Pkp2 Plakophilin 2 (predicted)1373743_at 3.2 RGD1305062 Similar to open reading frame 5 (predicted)1374518_at 3.1 LOC362011 Hypothetical protein LOC3620111374847_at 3.1 Lztr1 predicted Leucine-zipper-like transcriptional regulator, 1 (predicted)1368674_at 3.1 Pygl Liver glycogen phosphorylase1372297_at 3.1 Gsta4 Glutathione S-transferase, a 4 (predicted)1372752_at 3.1 Tm4sf7 Transmembrane 4 superfamily member 7 (predicted)1390131_at 3.0 Srr Serine racemase1374678_at 3.0 – Sema domain, immunoglobulin domain (Ig), transmembrane domain (TM)

and short cytoplasmic domain, (semaphorin) 4B1374512_at 3.0 Cdh7 Cadherin 7, type 21389412_at 3.0 – –1399071_at 3.0 – –1369909_s_at 3.0 Tm6p1 Fasting-inducible integral membrane protein TM6P11368774_a_at 3.0 Espn Espin1398249_at 2.9 Slc25a20 Solute carrier family 25 (carnitine/acylcarnitine translocase), member 201372895_at 2.9 RGD1309676 Similar to RIKEN cDNA 5730469M10 (predicted)1388555_at 2.8 Txnl5 Thioredoxin-like 5 (predicted)1372177_at 2.8 RGD:1359477 Molybdopterin synthase1372132_at 2.8 Cndp2 CNDP dipeptidase 2 (metallopeptidase M20 family) predicted1373337_at 2.7 Grhpr Glyoxylate reductase/hydroxypyruvate reductase (predicted)1372310_at 2.7 RGD1307632 Similar to tumor-related protein (predicted)1368331_at 2.7 Ctbs Chitobiase, di-N-acetyl-1386904_a_at 2.7 Cyb5 Cytochrome b-51373866_at 2.7 RGD:1359509 Similar to hypothetical protein FLJ134481368100_at 2.7 Pcyt2 Phosphate cytidylyltransferase 2, ethanolamine1368409_at 2.7 Gstt2 Glutathione S-transferase, u21390149_at 2.7 RGD:1303004 Transforming acidic coiled coil 2







1368355_at 2.7 Myo5b Myosin 5B1389552_at 2.7 – LIM and senescent cell antigen-like domains 1 (predicted)1372087_at 2.6 Harpb64 Hypertrophic agonist responsive protein B641374915_at 2.6 – –1375209_at 2.6 – Oxysterol binding protein-like 11 (predicted)1368077_at 2.6 Fbp1 Fructose-1,6-biphosphatase 11387669_a_at 2.6 Ephx1 Epoxide hydrolase 11383912_at 2.6 – cDNA clone MGC:109201 IMAGE:73067441376595_at 2.6 LOC500006 Similar to peroxisome biogenesis factor 11368071_at 2.6 Mg87 Mg87 protein1367793_at 2.6 Ddt D-Dopachrome tautomerase1368266_at 2.5 Arg1 Arginase 11373162_at 2.5 – Similar to hypothetical protein1385561_at 2.5 LOC500006 Similar to peroxisome biogenesis factor 11389074_at 2.5 Fchsd2 FCH and double SH3 domains 2 (predicted)1376265_at 2.4 – –1370321_at 2.4 Pdcd8 Programmed cell death 81392604_at 2.4 Nsdhl NAD(P) dependent steroid dehydrogenase-like1388755_at 2.4 Sec23a SEC23A (S. cerevisiae) (predicted)1367929_at 2.4 Cd59 CD59 antigen1371875_at 2.4 Manba Mannosidase, b A, lysosomal (predicted)1389394_at 2.4 – –1387357_at 2.4 Tmlhe Trimethyllysine hydroxylase, ´

1388788_at 2.4 Gcdh Glutaryl-coenzyme A dehydrogenase (predicted)1377096_at 2.4 RGD1309462 Similar to RIKEN cDNA 2310020P08 (predicted)1372510_at 2.3 – Neoplastic progression 31390387_at 2.3 – Similar to SH3 domain protein D19 (predicted)1374584_at 2.3 LOC361092 Similar to serine/threonine protein kinase 241387636_a_at 2.3 RGD:621692 P11 protein1386927_at 2.3 Cpt2 Carnitine palmitoyltransferase 21369629_at 2.3 Adk Adenosine kinase1367939_at 2.3 Rbp1 Retinol binding protein 1, cellular1377145_at 2.3 LOC294614 Similar to very large G protein-coupled receptor 11367702_at 2.3 Acadm Acetyl-coenzyme A dehydrogenase, medium chain1371919_at 2.3 RGD:1302935 Similar to RP2 protein, testosterone-regulated ricefield mouse (Mus caroli)1398384_at 2.3 Exosc9 Exosome component 9 (predicted)1368253_at 2.3 Gamt Guanidinoacetate methyltransferase1383080_at 2.3 Lamp2 Lysosomal membrane glycoprotein 21369660_at 2.3 Defb1 Defensin b 11369560_at 2.2 Gpd1 Glycerol-3-phosphate dehydrogenase 1 (soluble)1390378_at 2.2 – –1367773_at 2.2 Slc25a1 Solute carrier family 25, member 11372136_at 2.2 RGD:1305714 Similar to tetraspanin similar to TM4SF9 (predicted)1388358_at 2.2 RGD:1303312 Electron-transfer-flavoprotein, b polypeptide1374568_at 2.2 – Similar to mKIAA1737 protein (predicted)1371519_at 2.2 Etfdh Electron-transferring-flavoprotein dehydrogenase1373198_at 2.2 RGD1311098 Similar to RIKEN cDNA 2810451A06 (predicted)1367819_at 2.2 Got2 Glutamate oxaloacetate transaminase 21389642_at 2.2 – –1386466_at 2.2 – –1373335_at 2.2 LOC302808 Similar to zinc finger, DHHC domain containing 91370821_at 2.2 – Cb1-8121377357_at 2.1 – –1377601_at 2.1 RGD1311257 Similar to C21orf70 protein (predicted)1369023_at 2.1 Mipep Mitochondrial intermediate peptidase1369630_at 2.1 Adk Adenosine kinase1371560_at 2.1 Irf3 Interferon regulatory factor 31389345_at 2.1 – Transcribed locus, moderately similar to NP_862906.1 CD59b antigen

(Mus musculus)

Table A4 Probe sets downregulated by HNF4a2 only.


1377506_at 0.19 Gdf1 Growth differentiation factor 1 (predicted)1371165_a_at 0.24 Atp2a3 ATPase, Ca2q transporting, ubiquitous1368148_at 0.26 Ngfr Nerve growth factor receptor1371161_at 0.28 Ppp1r3b Protein phosphatase 1, regulatory (inhibitor) subunit 3B







1390250_x_at 0.28 – ATPase, class I, type 8B, member 21374236_at 0.31 – –1377772_at 0.32 Tmeff1 Transmembrane protein with EGF-like and two follistatin-like domains 11372537_at 0.32 – Echinoderm microtubule associated protein like 11373710_at 0.33 Slc38a4 Amino acid transport system A31370265_at 0.33 Arrb2 Arrestin, b 21390640_at 0.34 Chtf18 CTF18, chromosome transmission fidelity factor 18 homolog (S. cerevisiae)

(predicted)1378367_at 0.34 – –1392941_at 0.35 – –1368970_at 0.35 Cdh23 Cadherin 23 (otocadherin)1373223_at 0.35 – –1373977_at 0.35 – Kinesin family member 5C (predicted)1372624_at 0.36 – Transmembrane protein 16F (predicted)1373751_at 0.36 – –1379380_at 0.37 Spry1 Sprouty homolog 1 (Drosophila) (predicted)1375206_at 0.37 RGD1308212 Hypothetical LOC287466 (predicted)1374976_a_at 0.38 Soat1 Sterol O-acyltransferase 11388963_at 0.38 Asfn1 Astrotactin 11372637_at 0.38 – –1389132_at 0.39 Hip1 Huntingtin interacting protein 11373363_at 0.39 Map1b Microtubule-associated protein 1b1368104_at 0.39 Tspan2 Tetraspan 21383075_at 0.40 Ccnd1 Cyclin D11372182_at 0.40 Pfkp Phosphofructokinase, platelet1373399_at 0.40 Wdr6 WD repeat domain 6 (predicted)1390082_at 0.41 – –1370059_at 0.41 RGD:621458 Neurofilament, light polypeptide1370823_at 0.42 Bambi BMP and activin membrane-bound inhibitor, homolog (Xenopus laevis)1388464_at 0.42 – –1369004_at 0.43 Rab26 RAB26, member RAS oncogene family1370201_at 0.43 Calb1 Calbindin 11374575_at 0.43 RGD:1359613 cAMP responsive element binding protein 3-like 11372084_at 0.44 Ptp4a3 Protein tyrosine phosphatase 4a3 (predicted)1370367_at 0.44 Slc1a1 Solute carrier family 1 (neuronal/epithelial high affinity glutamate transporter,

system Xag), member 11388666_at 0.44 RGD:1303152 Ectodermal-neural cortex 11367874_at 0.45 Rhoq ras homolog gene family, member Q1376215_at 0.45 LOC499558 Similar to hypothetical protein 9630025C221369358_a_at 0.46 Hap1 Huntingtin-associated protein 11368404_at 0.46 Dbn1 Drebrin 11388193_at 0.46 Hip1 Huntingtin interacting protein 11370341_at 0.47 Eno2 Enolase 2, g

1371568_at 0.47 – –1369777_a_at 0.48 Shank2 SH3/ankyrin domain gene 2

Table A5 Probe sets upregulated by HNF4a8 only.


1389645_at 11.4 Prodh2 Proline dehydrogenase (oxidase) 2 (predicted)1387165_at 8.5 Maf v-maf musculoaponeurotic fibrosarcoma (avian) oncogene homolog (c-maf)1375924_at 5.8 – –1367598_at 4.9 Ttr Transthyretin1389943_at 4.2 Crocc predicted Ciliary rootlet coiled-coil, rootletin (predicted)1390472_at 3.9 – –1370950_at 3.5 Ppap2b ER transmembrane protein Dri 421368790_at 3.2 Serpina10 Serine (or cysteine) proteinase inhibitor, clade A (a-1 antiproteinase,

antitrypsin), member 101386938_at 3.2 Anpep Alanyl (membrane) aminopeptidase1370032_at 3.0 Slc9a3r1 ERM-binding phosphoprotein1375532_at 2.9 Id2 Inhibitor of DNA binding 2, dominant negative helix-loop-helix protein1383241_at 2.8 C1r Complement component 1, r subcomponent (predicted)1373480_at 2.5 – Heat shock 70 kDa protein 12A (predicted)1372523_at 2.4 Gclc Glutamate-cysteine ligase, catalytic subunit1372655_at 2.3 – –1388164_at 2.3 RT1.S3 RT1 class Ib, locus S31374620_at 2.3 Ceacam1 CEA-related cell adhesion molecule 1







1369048_at 2.3 Gabrd g-Aminobutyric acid A receptor, d subunit1368533_at 2.2 Heph Hephaestin1389474_at 2.2 – Myosin regulatory light chain interacting protein (predicted)1390366_at 2.2 LOC291002 Similar to PC-LKC gene product1387889_at 2.2 Folr1 Folate receptor 1 (adult)1387316_at 2.1 Cxcl1 Chemokine (C-X-C motif) ligand 11388153_at 2.1 Acsl1 Acyl-CoA synthetase long-chain family member 11374778_at 2.0 – –1388611_at 1.8 Tcea3 predicted Transcription elongation factor A (SII), 3 (predicted)

Table A6 Probe sets downregulated by HNF4a8 only.


1374160_at 0.47 – Similar to RIKEN cDNA 1110063G11 (predicted)1376966_at 0.46 – –1373152_at 0.44 RGD:1359545 Protease, serine, 231371692_at 0.43 RGD:1305525 Similar to AF1q (predicted)1387908_at 0.41 Rasd1 RAS, dexamethasone-induced 11370097_a_at 0.28 Cxcr4 Chemokine (C-X-C motif) receptor 41390403_at 0.24 RGD1304790 Similar to CG8312-PA (predicted)

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Received June 4, 2006; accepted September 8, 2006



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