(Survivin)-Knockout Mouse Model during Liver ... - ACS Publications

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Proteome Analysis of a Hepatocyte-Specific BIRC5 (Survivin)Knockout Mouse Model during Liver Regeneration Thilo Bracht,*,† Sascha Hagemann,‡ Marius Loscha,† Dominik A. Megger,† Juliet Padden,† Martin Eisenacher,† Katja Kuhlmann,† Helmut E. Meyer,†,§ Hideo A. Baba,‡,# and Barbara Sitek*,†,# †

Medizinisches Proteom-Center, Ruhr Universität Bochum, Bochum, Germany Institut für Pathologie, Universität Duisburg-Essen, Essen, Germany § Leibniz-Institut für Analytische Wissenschaften-ISAS-e.V, Dortmund, Germany ‡

S Supporting Information *

ABSTRACT: The Baculoviral IAP repeat-containing protein 5 (BIRC5), also known as inhibitor of apoptosis protein survivin, is a member of the chromosomal passenger complex and a key player in mitosis. To investigate the function of BIRC5 in liver regeneration, we analyzed a hepatocyte-specific BIRC5-knockout mouse model using a quantitative label-free proteomics approach. Here, we present the analyses of the proteome changes in hepatocyte-specific BIRC5-knockout mice compared to wildtype mice, as well as proteome changes during liver regeneration induced by partial hepatectomy in wildtype mice and mice lacking hepatic BIRC5, respectively. The BIRC5-knockout mice showed an extensive overexpression of proteins related to cellular maintenance, organization and protein synthesis. Key regulators of cell growth, transcription and translation MTOR and STAT1/STAT2 were found to be overexpressed. During liver regeneration proteome changes representing a response to the mitotic stimulus were detected in wildtype mice. Mainly proteins corresponding to proliferation, cell cycle and cytokinesis were up-regulated. The hepatocyte-specific BIRC5-knockout mice showed impaired liver regeneration, which had severe consequences on the proteome level. However, several proteins with function in mitosis were found to be up-regulated upon the proliferative stimulus. Our results show that the E3 ubiquitin-protein ligase UHRF1 is strongly up-regulated during liver regeneration independently of BIRC5. KEYWORDS: liver regeneration, survivin, E3 ubiquitin-protein ligase UHRF1, subcellular fractionation

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INTRODUCTION The Baculoviral IAP repeat-containing protein 5 (BIRC5), also known as inhibitor of apoptosis protein survivin, is a member of the family of inhibitor of apoptosis proteins (IAPs).1 Initially, BIRC5 was described for its capability to interfere with apoptosis2 but has later also been shown to play a central role in cell proliferation.3,4 BIRC5 fulfills crucial functions during mitosis and is part of the chromosomal passenger complex (CPC), a key player in mitosis and cytokinesis.5 The CPC consists of three nonenzymatic components, BIRC5, borealin and the inner centromere protein (INCENP), which facilitate and regulate the activity of the enzymatic component, the aurora kinase B.6 BIRC5 binds to INCENP, which functions as a scaffold for the other components of the CPC.7 During mitosis the CPC undergoes dynamic changes in localization, allowing specific phosphorylation of various substrates by aurora kinase B. The different localizations are related to the various functions of the complex, such as regulation of kinetochore-microtubule attachments and activation of the mitotic spindle assembly checkpoint.8,9 As a mediator of cytoskeletal and chromosomal events the CPC ensures correct separation of sister chromatids.10 © 2014 American Chemical Society

BIRC5 interacts with phosphorylated histone H3, thereby targeting the CPC to the inner centromere during early mitosis.11,12 The importance of BIRC5 for CPC function is evident since cells that lack BIRC5 are unable to complete mitosis and cell division and a global knockout of BIRC5 has shown to be lethal.5 BIRC5 attracted great attention because unlike other IAPs it is usually not or only weakly expressed in differentiated adult tissue but in contrast shows high expression in many kinds of cancer.13 BIRC5 expression has been shown to be correlated with tumor progression and a poor prognosis,14,15 which led to the proposal of BIRC5 as a potential drug target in cancer treatment.16 However, BIRC5 expression not only is limited to embryonic and malignant tissues but also is detected during proliferation, as reported for hematopoietic progenitor cells, vascular endothelial cells and several others.17−19 Expression of BIRC5 can also be observed during liver regeneration, as it has been shown for rodents after partial hepatectomy as well as in humans after split Received: December 3, 2013 Published: May 12, 2014 2771

dx.doi.org/10.1021/pr401188r | J. Proteome Res. 2014, 13, 2771−2782

Journal of Proteome Research

Article

Figure 1. Schematic presentation of the experimental design and the applied workflow.

liver transplantation.20 Although the function of BIRC5 has been extensively studied in various malignant diseases, its role in nontumor cell proliferation has not been investigated in great detail. Recently, we examined the impact of BIRC5 on mitosis in nontumor cells in vivo, using a conditional hepatocyte-specific BIRC5-knockout mouse model.21 Mice lacking hepatic BIRC5 showed a generally normal liver architecture but exhibited a decreased number of enlarged hepatocytes containing big nuclei with frequent vacuoles. After partial hepatectomy the liver regeneration in the BIRC5-knockout mice was impaired due to a decreased proliferative activity. The components of the CPC were not colocalized in the knockout mice, but INCENP and aurora kinase B were detected within intranucleic vacuoles. Substrates of aurora kinase B like histone H3 and histone H3-like centromeric protein A (CENPA) were not phosphorylated in the knockout condition, implicating that aurora kinase B activity was also impaired by the lack of BIRC5. Due to the comprehensive functional and histological impact of BIRC5 ablation, we decided to further analyze the molecular consequences of the BIRC5knockout in a global manner. Here we present a label-free LC−MS-based proteomics study, investigating our hepatocyte-specific BIRC5-knockout mouse model, before and 3 days after partial hepatectomy (PHE), respectively (see Figure 1). The aim of the study was to enlighten proteome changes related to the conspicuous phenotype of mice lacking hepatic BIRC5. Furthermore, we studied modulations of the proteome occurring during liver regeneration in wildtype mice compared to BIRC5-knockout mice using partial hepatectomy as a model for liver regeneration. We performed cell-compartment-specific sample fractionation before LC−MS analysis to increase the coverage of the analyzed proteome and to enrich nuclear proteins that we expected to be strongly affected by BIRC5. Differentially expressed proteins were functionally annotated and clustered using Ingenuity Pathway Analysis software. To verify the detected proteome changes, selected differentially expressed proteins were studied by Western blot analysis. By examining the influence of BIRC5 ablation on the liver proteome as well as proteome changes induced by partial hepatectomy, several key regulators of cell growth and proliferation were found to be affected. We describe the proteome changes related to liver regeneration induced by partial hepatectomy in wildtype mice. Here, we found that liver

regeneration is massively disturbed by the BIRC5-knockout, resulting in only minor proteome changes in mice lacking hepatic BIRC5. However, the conspicuous up-regulation of E3 ubiquitinprotein ligase UHRF1 during liver regeneration was found to be independent of the BIRC5 ablation.

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EXPERIMENTAL PROCEDURES

Animals and 70% Liver Resection

The generation of the investigated hepatocyte-specific BIRC5knockout mice was described recently.21 Seventy percent liver resection was carried out by a trained microsurgeon under intraperitoneally administered ketamine/xylazine anesthesia (0.6−1.2 mg ketamine/g bodyweight; 0.1−0.2 mg xylazine/g bodyweight). Animals were sacrificed 3 days after surgery, and whole livers were removed and used for further processing. All studies were performed with approval of the local Animal Ethics Committee. Experimental Design

For the label-free proteome analysis, 17 mice were used composed of 5 individual wildtype mice for each condition before (WTprePHE) and after hepatectomy (WTpostPHE), four BIRC5-knockout mice (KOprePHE) for the prehepatectomy condition, and three individuals after partial hepatectomy (KOpostPHE). For the immunological verification a new set of animal experiments was carried out and a total of 20 mice, 5 mice representing each condition, were analyzed. For the LC−MS/ MS analysis the samples were prefractionated by subcellular protein fractionation to increase the coverage of the analyzed proteome. All samples corresponding to a subcellular fraction were analyzed in an individual label-free study. Finally the results of the four different studies were compared and integrated manually. Subcellular Protein Fractionation

The subcellular fractions were generated using the Subcellular Protein Fractionation Kit for Tissue (Thermo Scientific, Bremen, Germany) according to the manufacturer’s instructions. Briefly, around 200 mg of liver tissue was chopped and subsequently homogenized in a Dounce homogenizer. The samples were resolved in protein-extraction buffer specific for cytosolic proteins. The solution was filtered through a tissue strainer and centrifuged at 500 × g for 5 min. The supernatant containing 2772



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peptide identifications were exported with the protein grouping function disabled. The identifications were matched to the corresponding LC−MS features, which were in parallel quantified using Progenesis LC−MS software (version 4.1.4804.41555, Nonlinear Dynamics Ltd., Newcastle upon Tyne, U.K.). Raw files were imported, and one run was automatically chosen by the software to match the features of the other runs of a particular experiment. A list of all features including m/z values and retention times was generated. Features with charges deviating from 2+, 3+ and 4+ were not considered for the analysis. The raw abundances of each feature were then automatically normalized to correct experimental variations (for detailed information on this step see ref 23). Only peptides unique for one protein in the respective experiment were used for quantification. The protein grouping option was disabled in data analysis with Progenesis LC−MS. Finally, quantitative results were statistically evaluated by one-way ANOVA. Proteins quantified with at least two peptides, an ANOVA p-value 2 were considered to be significantly differently expressed between the experimental groups.

cytosolic proteins was collected, and the protein pellet was dissolved in the next protein-specific extraction buffer. In total, the procedure was sequentially repeated 4 times using varying subcellular fraction-specific buffers and centrifugation speeds. Four different fractions were collected, containing cytosolic proteins, nuclear soluble proteins, nuclear chromatin-bound proteins, and membrane proteins. The fractionation efficiency was tested using Western blot analysis of the different sample fractions (Supplementary Figure 1). Sample Preparation and Tryptic Digestion

The protein concentration of the fractionated samples was determined using the Bradford assay (Bio-Rad, Hercules, CA, USA), and 5 μg of protein was loaded on a 18% Tris-glycine acrylamide gel (Anamed Electrophorese, Groß-Bieberau, Germany). The samples were allowed to run slightly into the gel (15 min, 100 V) and form a single band of approximately 3 mm height. The bands were stained with Coomassie, manually cut from the gel, and digested with trypsin (Serva Electrophoresis, Heidelberg, Germany) in 10 mM ammonium bicarbonate buffer (pH 7.8) overnight at 37 °C.22 Tryptic peptides were extracted with 20 μL of 50% acetonitrile in 0.1% TFA (1:1) by sonication (15 min, on ice) for two times. Subsequently, acetonitrile was removed by vacuum centrifugation. The peptide concentration was determined by amino acid analysis (AAA) as described earlier.23

Functional Annotation of Regulated/Differentially Expressed Proteins

The Ingenuity Pathway Analysis software (IPA, Version 12402621, Ingenuity Systems, Redwood City, CA, USA) was used to annotate the protein functions and localizations. The tool was also used to cluster the differentially expressed proteins regarding their functions in networks and canonical pathways, respectively. Therefore, UniProtKB accessions as well as fold change values and ANOVA p-values were imported into IPA. A core analysis was performed using the Ingenuity knowledge base as reference set. Direct and indirect relations and all data sources were considered. Only experimentally observed relations were allowed. Taxonomy was restricted to mice, and data derived from primary cells and the liver were considered.

LC−MS/MS Analysis

The label-free proteomics study was performed with slight modifications according to a workflow that has recently been described.24 The RPLC−MS/MS analysis was performed using an Ultimate 3000 RSLCnano system (Thermo Scientific, Bremen, Germany) online coupled to an Orbitrap Elite mass spectrometer (Thermo Scientific, Bremen, Germany). The injection volume was 15 μL of the sample, representing 250 ng of peptides. After injection the samples were preconcentrated with 0.1% TFA on a trap column for 7 min (Acclaim PepMap 100, 300 μm × 5 mm, C18, 5 μm, 100 Å; flow rate 30 μL/min). Subsequently, the peptides were transferred to the analytical column (Acclaim PepMap RSLC, 75 μm × 50 cm, nano Viper, C18, 2 μm, 100 Å) and separated by a gradient from 5% to 40% solvent B over 98 min (solvent A: 0.1% FA, solvent B: 0.1% FA, 84% acetonitrile; flow rate 400 nL/min; column oven temperature 60 °C). The MS was operated in a data-dependent mode. Full scan MS spectra were acquired at a resolution of 60,000 in the Orbitrap analyzer, while tandem mass spectra of the 20 most abundant peaks were measured in the linear ion trap after peptide fragmentation by collision-induced dissociation (CID).

Western Blot Analysis

Liver samples were lysed by sonication in urea sample buffer as previously described,23 and the protein concentration was determined using a Bradford assay. Equal amounts of 15 μg of protein per sample were loaded and electrophoretically separated on a 4−20% polyacrylamide gel (Criterion TGX Stain-free, BioRad, Hercules, CA, USA). Gels were exposed to UV light for 1 min to activate the trihalo compounds contained in the gel, which react with tryptophan residues to enable visualization of the proteins. The proteins were transferred to a nitrocellulose membrane (Trans-Blot Turbo, Bio-Rad, Hercules, CA, USA), and unspecific binding sites were blocked for 30 min at room temperature using StartingBlock blocking solution (Thermo Scientific, Bremen, Germany). Primary antibodies were incubated overnight at 4 °C in 50% StartingBlock in TBST (50 mM Tris-HCl, 150 mM NaCl, pH 7.4, 0.1% Tween-20). For the primary antibody dilutions that were used, see Table 1. Membranes were washed with TBST and incubated with horseradish peroxidase-labeled secondary antibodies (Jackson ImmunoResearch, Newmarket, U.K.) in the same buffer for 1 h at room temperature. Membranes were washed again using TBST. For documentation the ChemiDoc MP Imaging system was used (Bio-Rad, Hercules, CA, USA). Bound antibodies were visualized by enhanced chemiluminescence (GE Healthcare, Munich, Germany). The total protein amount on the membranes was detected using stain-free visualization. Pictures were analyzed with Image Lab software (Bio-Rad, Hercules, CA, USA). Densitometric analysis was performed, and bands were

Peptide Identification and Quantification

The protein and peptide identification was performed for all LC−MS/MS runs using Proteome Discoverer (ver. 1.3.0.339, Thermo Fisher Scientific, Rockford IL, USA) searching against UniProtKB/Swiss-Prot database (Uniprot/Swissprot Release 2012_09, 538,010 entries) using Mascot (version 2.3.0.2.).25 The following search parameters were applied: mass tolerances were set to 5 ppm and 0.4 Da for precursor and fragment ions, respectively. Taxonomy was restricted to Mus musculus, and one enzymatic miscleavage was allowed. Dynamic modifications of cysteine (propionamide) and methionine (oxidation) were considered. The confidence of peptide identifications was estimated using the percolator function, implemented in Proteome Discoverer. Peptide identifications with false discovery rates >1% (q-value >0.01) were discarded. For quantification the 2773



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Table 1. Primary Antibodies Used for Verificationa antibody

distributor

product ID

source

dilutionb

anti-UHRF1 anti-PCNA anti-STAT1 anti-MTOR anti-MCM5 anti-EF2 anti-KIF4 anti-FLOT1

Santa Cruz GeneTex abcam Cell Signaling abcam abcam GeneTex abcam

sc-98 817 GTX100 539 ab2415 #2972 ab17 967 ab33 523 GTX63 429 ab41 927

rabbit rabbit rabbit rabbit rabbit rabbit rabbit rabbit

1:1000 1:500 1:1000 1:1000 1:2000 1:2000 1:5000 1:1000

a

The differential expression between experimental groups was methodologically verified using Western blot analysis for eight selected proteins. bDilutions of primary antibodies for incubation.

normalized to total protein content of the respective lanes. The relative band volume was calculated, and the lane measurements were tested for significance using Student’s t test (dependent on experimental setup paired or unpaired, two-sided, unequal variances).

Figure 2. Efficiency of the applied subcellular fractionation. Localizations of proteins identified in the different fractions. Treatment of liver samples led to an enrichment of cytosolic proteins to 61% of all proteins identified in the respective fraction. For both nuclear fractions 27% and 29%, respectively, of all proteins were annotaded for nuclear localization. The enrichment of membrane proteins resulted in 13% of proteins annotated for plasma membrane localization.

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RESULTS In order to gain new insights into the function of BIRC5 during liver regeneration, we analyzed a hepatocyte-specific BIRC5knockout mouse model. The mice were subjected to partial hepatectomy (PHE) to induce liver regeneration, and the livers were analyzed prior to and after PHE. To increase the coverage of the proteome under investigation and to enrich especially nuclear proteins, we carried out a subcellular protein fractionation of the samples. For each subcellular fraction we performed a discrete label-free proteomics study.

proteins were assigned to features by Progenesis LC−MS software and considered for quantification. For the soluble nuclear fraction 2928 proteins (2554 protein groups, respectively) were identified, and 2289 proteins were assigned to quantified features. One LC−MS/MS run was discarded for technical reasons. Thus, a total of 16 samples were analyzed (5 × WTpreHEP, 4 × KOpreHEP, 5 × WTpostHEP, 2 × KOpostHEP). For the nuclear chromatin-bound fraction 2007 proteins (1725 protein groups) could be identified of which 1547 were assigned to features. Two runs had to be excluded from analysis, resulting in 15 samples included in the analysis (3 × WTpreHEP, 4 × KOpreHEP, 5 × WTpostHEP, 3 × KOpostHEP). For the membrane fraction, 2651 proteins (2226 protein groups) were identified, and 1717 proteins were assigned to features. One LC−MS/MS measurement was excluded, and 16 samples could be analyzed (5 × WTpreHEP, 4 × KOpreHEP, 5 × WTpostHEP, 2 × KOpostHEP). In all fractions together in total 4348 proteins (corresponding to 3799 protein groups) were identified. The data have been deposited to the ProteomeXchange with identifier PXD000625. For each of the four label-free proteomics studies four different experimental designs were applied. The following combinations were tested for significantly differential abundances of proteins between the experimental groups: the livers of wildtype mice were compared to livers of mice lacking hepatic BIRC5 before treatment by partial hepatectomy to detect proteome changes in the BIRC5-knockout condition (WTprePHE vs KOpreHEP). For both wildtype and BIRC5-knockout mice, samples from livers before and after partial hepatectomy were analyzed (WTprePHE vs WTpostPHE; KOprePHE vs KOpostPHE). Finally, liver samples from wildtype mice and BIRC5-knockout mice were also compared after partial hepatectomy (WTpostPHE vs KOpostPHE).

Subcellular Protein Fractionation

Efficiency of subcellular fractionation was tested using Western blot analysis. Staining of marker proteins showed a satisfying enrichment of marker proteins in the corresponding fractions (Supplementary Figure 1). The proteins identified in each fraction were annotated regarding their subcellular locations (Figure 2). In the cytosolic fraction 1741 proteins, corresponding to 61% of all proteins identified in this fraction, were annotated for a cytosolic localization, and 774 proteins of the soluble nuclear fraction were annotated for nuclear localization. The percentage of nuclear proteins was increased to 27% in relation to the other subcellular fractions. For the nuclear chromatin bound fraction 578 proteins, corresponding to 29%, were annotated for nuclear localization. A moderate increase to 13% of membrane proteins (342 proteins) was observed in the membrane fraction. However, 91 membrane proteins were exclusively identified in this fraction. Quantitative Label-Free Proteomics Analysis

We performed individual label-free proteomics studies for each subcellular protein fraction. For the quantitative label-free analysis several samples had to be excluded from analysis due to technical problems during the LC−MS/MS measurements. The runs did not fit our quality criteria as the alignment of the runs during the quantitative analysis with Progenesis LC−MS was not possible. Because of limited sample material it was not possible to repeat the LC−MS/MS analysis of these samples. In the cytosolic fraction a total number of 2919 proteins (corresponding to 2546 protein groups in proteome discoverer) was identified. Two runs had to be excluded for technical reasons, which resulted in the analysis of 15 samples (4 × WTpreHEP, 4 × KOpreHEP, 4 × WTpostHEP, 3 × KOpostHEP). 2195

Differentially Expressed Proteins

By comparing livers of wildtype and hepatocyte-specific BIRC5knockout mice (WTprePHE vs KOprePHE) 762 proteins were found to be significantly differentially expressed between both 2774



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Figure 3. Functional annotation of differentially expressed proteins. Proteins showing significant differences in expression between the experimental conditions were annotated for their molecular and cellular functions using Ingenuity Pathway Analysis. (A) Hepatocyte-specific BIRC5-knockout mice vs wildtype mice prior to partial hepatectomy. (B) Protein regulation upon partial hepatectomy in wildtype mice. (C) Hepatocyte-specific BIRC5knockout mice vs wildtype mice after partial hepatectomy. Blue bars indicate the number of proteins annotated with the corresponding function. Red bars indicate the respective p-values.

rejected from further analysis. By comparing BIRC5-knockout mouse livers before and after partial hepatectomy (KOprePHE vs KOpostPHE), 49 significantly differential proteins were detected (Supplementary Table 3). Twenty-one proteins were up-regulated and 28 proteins were down-regulated as a result of partial hepatectomy. The comparison of BIRC5-knockout and wildtype mouse livers after partial hepatectomy (WTpostPHE vs KOpostPHE) revealed a significant differential expression of 203 proteins. In the BIRC5-knockout condition 123 proteins showed elevated expression levels, whereas 80 proteins showed a higher expression in the wildtype mice (Supplementary Table 4).

conditions (Supplementary Table 1). 529 proteins were overexpressed in livers of BIRC5-knockout mice, and 223 proteins showed a lower expression level in the knockout condition. Ten proteins were found to have conflicting results in different subcellular fractions. These proteins were excluded from the subsequent analysis. The investigation of proteome changes in wildtype mouse livers during liver regeneration (WTprePHE vs WTpostPHE) revealed 543 proteins to be significantly regulated (Supplementary Table 2). After partial hepatectomy 341 proteins appeared to be up-regulated, and 192 proteins down-regulated. Ten proteins showed inconsistent regulation directions in different subcellular fractions and were 2775



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Table 2. Differentially Expressed Proteins Used for Verification gene name

experimental setupa

UHRF1

WTprePHE vs WTpostPHE KOprePHE vs KOpostPHE

PCNA

WTprePHE vs WTpostPHE

STAT1

KOprePHE vs KOpostPHE WTprePHE vs KOprePHE WTpostPHE vs KOpostPHE

MTOR

WTprePHE vs WTpostPHE WTprePHE vs KOprePHE

MCM5

WTprePHE vs WTpostPHE WTprePHE vs KOprePHE

EF2 KIF4 FLOT1

WTprePHE vs WTpostPHE WTprePHE vs WTpostPHE KOprePHE vs KOpostPHE WTprePHE vs KOprePHE

subcellular fractionb nuclear soluble fraction nuclear chromatin-bound fraction nuclear chromatin-bound fraction cytosolic fraction nuclear soluble fraction cytosolic fraction nuclear soluble fraction cytosolic fraction membrane fraction membrane fraction cytosolic fraction nuclear soluble fraction cytosolic fraction nuclear soluble fraction cytosolic fraction nuclear soluble fraction nuclear soluble fraction nuclear soluble fraction nuclear soluble fraction

peptides used for quantification

ANOVA p-value

fold change

highest mean condition

2 3

0.011 0.01

81.45 136.23

WTpost PHE KOpost PHE

4

0.015

27.05

WTpost PHE

2 5 11 5 11 2 2 13 4 6 4 6 29 8 2 2

0.039 0.003 0.01 0.001 0.019 0.006 0.0001 0.004 0.022 0.041 0.018 0.004 0.045 0.003 0.04 0.048

2.42 5.29 3.11 3.91 3.94 2.64 3.12 5.72 5.43 8.93 3.05 3.71 2.4 12.40 2.72 13.69

KOpost PHE KOprePHE KOprePHE KOpostPHE KOpostPHE WTpostPHE KOprePHE KOprePHE WTpostPHE WTpostPHE KOprePHE KOprePHE WTpostPHE WTpostPHE KOprePHE KOprePHE

a

Four different experimental setups were statistically evaluated. Livers of wildtype mice (WT) and hepatocyte-specific BIRC5-knockout mice (KO) were analyzed prior to (prePHE) and 72 h after partial hepatectomy (postPHE). bIndividual label-free approaches were performed using four different subcellular fractions. The respective fraction indicates the label-free approach in which the proteins were found to be significantly differentially expressed.

Functional Interpretation of Differentially Expressed Proteins

such proteins are often involved in malignant diseases, the annotation of regulated proteins resulted in a large number of cancer-related proteins (Figure 3B). In detail, we could observe an up-regulation of MTOR and IEF signaling as well as proteins involved in regulation of translation (e.g., EF2), mitosis (e.g., KIF4) and DNA replication (e.g., PCNA). We found several proteins of the mini-chromosome maintenance complex (MCM complex) to be up-regulated upon PHE. A conspicuous finding was the massive up-regulation of E3 ubiquitin-protein ligase UHRF1, which was found to be 81.4-fold higher expressed after PHE (Table 2). The proteins that were found to be downregulated during liver regeneration were predominantly related to regular liver functions such as small molecule metabolism and energy production (Figure 3B). The hepatocyte-specific BIRC5-knockout has enormous consequences for liver regeneration on the proteome level. Only the relatively small number of 49 proteins was found to be significantly differentially expressed in mutant mice before and after PHE (compared to 543 in wildtype mice). This result clearly resembles the disruption of functional liver regeneration, which has already been observed under macroscopic conditions.21 However, 7 of 21 up-regulated proteins fulfill functions related to DNA replication and transcription (e.g., PCNA). Therefore, these proteins are likely to be influenced by regulatory mechanisms independent of BIRC5. The most striking finding was the strong up-regulation of E3 ubiquitin-protein ligase UHRF1 (Table 2), which was also found to be strongly upregulated in livers of wildtype mice. The comparison of the hepatocyte-specific BIRC5-knockout and wildtype livers after partial hepatectomy is mainly affected by the differences between both conditions that were already observed before the operative treatment. Primarily proteins involved in metabolic liver function were found to be underrepresented in the knockout condition (Figure 3 C). STAT1 and

Proteins with a significant differential expression were annotated regarding their molecular functions using the Ingenuity Pathway Analysis software. The likelihood that a set of proteins corresponds to a certain biological function was calculated using Fisher’s Exact Test. Additionally, proteins were reviewed manually for functional clusters, and results achieved by Ingenuity Pathway Analysis software were critically evaluated by literature research. The comparison of BIRC5-knockout and wildtype mouse livers prior to partial hepatectomy (WTprePHE vs KOprePHE) showed a lower expression of proteins associated with metabolic functions, such as energy production and metabolism of small molecules like lipids and amino acids in the BIRC5-knockout (Figure 3 A). This finding was substantiated by the observation that 60% of the proteins showing a lower expression level in the BIRC5-knockout condition were enzymes (Supplementary Figure 3). In contrast, proteins related to cellular maintenance and cellular assembly and organization were overexpressed in BIRC5-knockout mice livers. This included processes such as vesicle formation and Golgi trafficking. Regulators of actin cytoskeleton ROCK1 and ROCK2 (Rho-associated protein kinase 1 and 2, Supplementary Table 1) were overexpressed. We found a remarkable number of E3 ubiquitin ligases and proteins associated with ubiquitination to be overexpressed. Expression of MTOR (mammalian target of rapamycin) and proteins involved in IEF signaling as well as ribosomal proteins and MCM proteins was elevated and the activators of transcription STAT1 and STAT2 were found to be overexpressed in BIRC5-knockout livers (Table 2). The analysis of unbiased liver regeneration in wildtype mice (WTprePHE vs WTpostPHE) revealed massive up-regulation of proteins related to proliferation, cell cycle and cytokinesis. As 2776



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Figure 4. Immunological verification of selected proteins. Expression levels of eight selected proteins were investigated using Western blot analysis. Box plots show the results of densitometric evaluation (boxes represent 25th and 75th percentile, whiskers indicate the standard deviation, median is shown as a black bar, and the mean value as a square within boxes). The differences between experimental conditions were tested for statistical significance using Student’s t test (either paired or unpaired, two-sided, unequal variances).

STAT2 were found to be overexpressed, whereas STAT3 had a lower abundance in the knockout. Proteins associated with cellular organization and morphology were overexpressed in

livers of BIRC5-knockout mice. Naturally, proteins with function in proliferation, which were previously overexpressed in the knockout (e.g., MTOR, proteins related to IEF-signaling, MCM 2777



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reflect previous macro- and microscopic observations. While the wildtype livers predominantly showed an expression of proteins related to metabolism of the liver, an extensive overrepresentation of proteins related to cellular maintenance and intracellular transport was observed in the knockout condition (Figure 3A). In particular cytoskeletal proteins and regulators of cytoskeleton signaling, proteins involved in vesicular transport, protein folding and degradation were overexpressed in BIRC5deficient mice. These changes in expression levels are likely to be a result of the increased cell size and volume, which seem to afford an extensive cellular organization. We also found an overexpression of several proteins related to the transcription and translation machinery in the knockout, indicating an increased protein production and turnover, the latter supported by many E3 ubiquitin ligases that were found to be overexpressed in the knockout condition. MCM proteins and proteins involved in IEF-signaling were overexpressed prior to PHE, but were also found to be up-regulated during liver regeneration in wildtype mice. Notably key regulators of cell growth and transcription MTOR, STAT1 and STAT2 were overexpressed in the BIRC5knockout, indicating regulatory mechanisms that were affected by BIRC5 ablation. Mammalian target of rapamycin (MTOR) is a highly conserved serine/threonine kinase that has a fundamental function in the control of cell growth (i.e., increase in size and mass). It positively regulates anabolic processes such as transcription and protein synthesis.28 MTOR is part of two different complexes, called TORC1 and TORC2. The regulatory-associated protein of MTOR (Raptor), which is together with MTOR part of TORC1, was also found to be overexpressed in the knockout condition (fold change 8.54). While TORC1 drives cell growth by regulating transcription and translation, TORC2 regulates the organization of the actin cytoskeleton, therefore controlling the spatial aspects of cell growth. Both functions of the two TOR complexes correspond with the observed proteome changes in the knockout. The overexpression of MTOR is likely to contribute to the increased cell volume, which characterizes the phenotype of the hepatocyte-specific BIRC5-knockout. However, the connection between BIRC5 and MTOR remains to be clarified. Interestingly, BIRC5 expression was shown to be partially regulated by IGF-1/MTOR signaling.29 STAT (signal transducer and activator of transcription) proteins are activated by a variety of growth factors and cytokines. Upon activation they form dimers, which translocate into the nucleus where they regulate gene expression. The JAK (Janus kinase)-STAT signaling has initially been studied in the context of immunity and response to interferon, but STAT proteins are also known to be involved in many pathological processes in the liver.30,31 In liver injury and regeneration activation of STAT1 has been described to promote apoptosis and prevent hepatocyte proliferation as well as liver regeneration. In contrast, activation of STAT3 is protective and promotes liver regeneration.32−34 STAT1 has pro-inflammatory activity and leads to apoptosis of hepatocytes, whereas STAT3 functions as anti-inflammatory signal.33 Notably BIRC5 is a direct downstream target of STAT3.35 As STAT proteins are phosphorylated by JAK proteins, we have to note that we only measured protein abundances but not activation states. However, we observed increased levels of STAT1 and STAT2 in the BIRC5-knockout condition compared to the wildtype. As STAT1 is described to inhibit liver regeneration, an increased activation of STAT1 would be in line with our observations (impaired liver

proteins), were no more found to be differentially expressed, since they were up-regulated during liver regeneration in wildtype mice. Western Blot Analysis

In order to verify the differences in protein expression levels that were observed in the label-free proteomics approach, we performed Western blot analysis of eight selected proteins. Western blots were evaluated via densitometry (Figure 4). UHRF1 and PCNA were found in the label-free proteomics study to be up-regulated after PHE in both knockout and wildtype mice. This finding could be verified by Western blot analysis. For UHRF1 the regulation in the knockout condition was significant, whereas the regulation in the wildtype was not but was clearly visible on manual inspection. For PCNA the upregulation was significant for both conditions. STAT1 was found to be overexpressed in the knockout mice compared to wildtype mice before and after PHE. The differential expression was methodologically verified. The Western blot analysis also revealed an increase of STAT1 expression upon PHE in wildtype and knockout mice, which was not detected by the proteomics approach. MTOR was also found to be overexpressed in the knockout condition. This finding was substantiated by Western blot analysis. However, MTOR was also found to be up-regulated during liver regeneration in wildtype mice, which was apparent in the immunoblots by trend but was not significant. In the proteomics approach MCM5 was found to be up-regulated during liver regeneration in wildtype mice and overexpressed in knockout mice before PHE. The immunological verification confirmed these results and in addition revealed that MCM5 was also up-regulated upon PHE in the BIRC5-deficient mice. Upon PHE EF2 and KIF4 were found to be up-regulated in wildtype mice by the proteomics approach, which was successfully verified by Western blot analysis. For KIF4 the analysis also showed a much weaker up-regulation upon PHE in the BIRC5-deficient mice, which was statistically significant. For EF2 there was a trend observable that expression levels after PHE are also slightly lower in knockout mice than in the wildtype. In the proteomics approach FLOT1 was found to be overexpressed in knockout mice before PHE. This finding was confirmed by the verification experiments. Upon PHE, FLOT1 was also found to be downregulated in the knockout condition, which was observable by trend in the immunoblots but was not significant. An upregulation during liver regeneration in wildtype mice was not detected by the proteomics approach, but was highly significant in the immunological analysis.

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DISCUSSION The phenotype of mice lacking hepatic BIRC5, manifesting in a reduced cell number, increased cell volume, macronucleation and polyploidy, was reported earlier in two independent studies.21,26 A similar phenotype was also reported for a cardiomyocyte-specific knockout of BIRC5.27 A number of proteins and molecular consequences were recently described to be related to this conspicuous phenotype.21 However, we decided to further dissect the impact of BIRC5 ablation on the liver proteome and its changes during liver regeneration using a quantitative proteomics approach. Proteome Changes in the Hepatocyte-Specific BIRC5 Knockout Mouse Model

As expected we were able to observe massive differences between the proteomes of wildtype and BIRC5-knockout livers prior to partial hepatectomy. Our findings on the molecular level closely 2778



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responsible for ensuring proper regulation of anaphase spindle dynamics by phosphorylation of KIF4A.45 As aurora kinase B function is impaired in BIRC5-knockout mice,21 chromosome segregation is likely to be affected as well. While in unbiased liver regeneration up-regulation of KIF4 represents mitotic activity, the functional impairment of KIF4 in the knockout condition might lead to a rudimentary up-regulation upon PHE. Disturbed activity of KIF4 and therefore impaired chromosome segregation probably contributes to the described macronucleation and polyploidy of the BIRC5-knockout hepatocytes. Beside regulations related to DNA replication and mitosis also proteins involved in protein biosynthesis were found to be upregulated during liver regeneration. Elongation factor 2 (EF2), together with EF1A (Elongation factor 1-alpha 1), conducts the cycle of protein elongation. It is negatively regulated via phosphorylation of threonine 56 by EF2-kinase resulting in down-regulation of translation.46 EF2 was also shown to be increasingly phosphorylated during mitosis, when overall protein translation decreases.47 Upon regeneration after PHE we were able to detect up-regulation of EF2 in wildtype mice. Notably, MTOR signaling (MTOR also was found to be up-regulated during liver regeneration) is involved in the regulation of protein translation via EF2.46 As liver regeneration is a proliferative process it affords tight control of protein synthesis, up-regulation of EF2 seems to be a physiological reaction to the mitotic stimulus. It has to be pointed out that in our study only overall protein expression irrespective of phosphorylation states was examined. Therefore, activation status of EF2 during liver regeneration cannot be determined. The response to PHE differed strongly between BIRC5knockout mice and wildtype mice on the proteome level, since only a comparably small number of protein regulations induced by PHE could be detected. This, of course, resembles the macroscopic observations, as liver regeneration does not take place in the absence of hepatic BIRC5. Surprisingly, we were nevertheless able to detect a number of striking regulations associated with unbiased liver regeneration in mutant mice. Proliferating cell nuclear antigen (PCNA) was found to be upregulated after PHE in wildtype and BIRC5-knockout mice. PCNA acts as a sliding clamp for DNA polymerases and therefore is an important cofactor for DNA replication. It is involved in several crucial processes concerning DNA duplication, e.g., orchestrating replication events, repair processes, chromatin assembly and remodeling, cell cycle control and survival.48 PCNA is overexpressed in a multitude of cancers and has also been proposed as a drug target for treatment of malignancies.49 Notably, PCNA directly interacts with the E3 ubiquitin-protein ligase UHRF1, which was also found to be upregulated after PHE in both conditions, the unbiased liver regeneration in wildtype mice and the response to PHE in BIRC5-knockout mice. UHRF1, also known as nuclear zinc finger protein Np95 or ICBP90 in humans, plays a critical role in cell cycle progression. It is responsible for maintenance of DNA methylation status during DNA replication by interaction with hemimethylated DNA and recruitment of DNA (cytosine-5)methyltransferase 1 (DNMT1).50−52 During DNA replication UHRF1 directly interacts with PCNA. UHRF1 has been shown to be required for cell cycle progression in several cell culture models.53−55 It is expressed in various kinds of cancer including lung,56 breast57 and prostate cancer.58 The expression of UHRF1 in human fetal livers was observed on the transcript level.59 In zebrafish UHRF1 gene expression has been shown to be required for liver regeneration. The expression of UHRF1 mRNA was

regeneration). In contrast, a pro-apoptotic effect of STAT1 is contradictory, as increased apoptosis has not been observed in the knockout.21 After partial hepatectomy STAT3 was less abundant in knockout compared to wildtype mice, therefore suggesting a minor induction of proliferation, which also corresponds to the impaired liver regeneration in the knockout. Another interesting finding was the strong overexpression of the lipid raft protein Flotillin-1 (FLOT1) in the BIRC5knockout. FLOT1 is involved in several membrane-associated signaling pathways and additionally regulates actin cytoskeleton dynamics, clathrin-independent endocytic pathways and phagosome traffic.36−40 Furthermore, FLOT1 was shown to localize to the nucleus upon mitotic stimuli.41 It was found that a knockdown of FLOT1 resulted in effects similar to a depletion of aurora kinase B and a direct interaction of FLOT1 with aurora kinase B was demonstrated.42 A role of FLOT1 in determining the amount of aurora kinase B available to form the chromosomal passenger complex to exhibit its important functions in mitosis was suggested. The overexpression of FLOT1 could represent a consequence of impaired aurora kinase B activity, which has been described for BIRC5-knockout hepatocytes.21 The up-regulation of FLOT1 during liver regeneration in wildtype mice was detectable in Western blot and is likely to be caused by the mitotic stimulus. Surprisingly, in the knockout condition a slight down-regulation upon PHE was observed. In the proteomics approach FLOT1 was detected only in the nuclear soluble fraction, indicating a nuclear localization of the protein. Proteome Changes Induced by Partial Hepatectomy

In the wildtype mice partial hepatectomy induced massive changes of the proteome. We observed a massive up-regulation of proteins related to mitosis and cell proliferation of which some were also found to be overexpressed in the BIRC5 knockout. The MCM proteins 2, 3, 5, 6, and 7 were consistently found to be significantly up-regulated during liver regeneration in wildtype mice and overexpressed in BIRC5-knockout mice. The MCM proteins 2−7 form the minichromosome maintenance complex (MCM complex), which is responsible to ensure correct replication of the DNA during mitosis by licensing origin DNA before the S phase.43 Misregulation of this system is discussed in carcinogenesis and genomic instability. Reactivation of the licensing system during the S phase seems to end up in irreversible duplication of chromosomal segments. The observed up-regulation of the DNA licensing complex in wildtype livers after PHE can be assigned to the physiological DNA replication as a consequence of the mitotic stimulus. BIRC5-knockout livers already showed elevated levels of MCM5 prior to PHE, which might reflect the macronucleation and polyploidy of the hepatocytes in this model.21 Western blot analysis of MCM5 revealed that also in the knockout condition an up-regulation of MCM5 takes place upon PHE, showing that although cell proliferation is generally impaired, mechanisms related to DNA replication are induced. The chromosome-associated kinesin KIF4 was found to be upregulated during liver regeneration in wildtype mice by the proteomics approach. Western blot analysis identified a weaker up-regulation of KIF4 upon PHE also in BIRC5-knockout mice. KIF4 is involved in the control of the central spindle formation in anaphase, mitotic chromosomal positioning and bipolar spindle stabilization.44,45 Recently it was shown that aurora kinase B directly phosphorylates KIF4’s human homologue KIF4A, resulting in its localization to the central spindle to control microtubule polymerization. Aurora kinase B seems to be 2779



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German federal state North Rhine-Westphalia (NRW), project number z0911bt004e. A part of this study was funded by P.U.R.E. (Protein Unit for Research in Europe), a project of North Rhine-Westphalia, a federal state of Germany. The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral. proteomexchange.org) via the PRIDE partner repository with the data set identifier PXD000625 and DOI 10.6019/ PXD000625. The authors thank the PRIDE Team as well as Ulian Uszkoreit for their assistance during the data upload.

observed during liver regeneration after PHE in zebrafish and mice. This data indicates that UHRF1 is required for liver growth in developing embryonic livers and in regeneration.60 To our knowledge this is the first time that UHRF1 expression was shown to be elevated during liver regeneration on the protein level. Our findings clearly show that the up-regulation of UHRF1 during liver regeneration is independent of the presence of BIRC5.

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CONCLUSION Our study examined the proteome changes in a hepatocytespecific BIRC5-knockout mouse model, as well as the proteome changes that occur during liver regeneration induced by partial hepatectomy. This is the first time that a quantitative proteome analytical approach was used to shed light on the molecular basis of the conspicuous phenotype of hepatocytes lacking BIRC5. We found that key regulators of cell growth, transcription and translation MTOR and STAT1/STAT2 were overexpressed in mice lacking hepatic-BIRC5, a finding that is appropriate to explain the relation between proteome changes and the phenotype. Probably, our results could be partially transferable to other BIRC5-knockout models, which share a similar phenotype. We described the proteome changes in wildtype mice livers that occurred upon partial hepatectomy-induced liver regeneration and show that in hepatocyte-specific BIRC5knockout mice impaired liver regeneration also has severe consequences on the proteome level. Rather than identifying proteins that were compensatory regulated in this issue, we were able to identify proteins regulated independently of BIRC5, among them the E3 ubiquitin-protein ligase UHRF1, which is known to fulfill a central role in liver regeneration.

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ABBREVIATIONS PHE, partial hepatectomy; LC−MS/MS, liquid chromatography−tandem mass spectrometry; RPLC, reversed phase liquid chromatography; EIF, eukaryotic translation initiation factor; UHRF1, E3 ubiquitin-protein ligase UHRF1; PCNA, proliferating cell nuclear antigen; STAT1, signal transducer and activator of transcription 1; MTOR, serine/threonine-protein kinase mTOR; MCM5, DNA replication licensing factor MCM5; EF2, elongation factor 2; KIF4, chromosome-associated kinesin KIF4; FLOT1, flotilin-1

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ASSOCIATED CONTENT

S Supporting Information *

Tables of significant differentially expressed proteins WTprePHE vs KOprePHE, significant differentially expressed proteins WTprePHE vs WTpostPHE, significant differentially expressed proteins KOprePHE vs KOpostPHE, and significant differentially expressed proteins WTpostPHE vs KOpostPHE. Figures of Western blot analysis of subcellular fractionation efficiency, protein types of proteins significantly regulated upon partial hepatectomy, and protein types of proteins differentially expressed between hepatocyte-specific BIRC5-knockout mice and wildtype mice. This material is available free of charge via the Internet at http://pubs.acs.org.

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REFERENCES

AUTHOR INFORMATION

Corresponding Authors

*Tel. +49-(0)-234/32-29985. E-mail: [email protected]. *Tel. +49-(0)-234/32-24362. E-mail: [email protected]. Author Contributions #

These authors contributed equally to this work.

Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS The authors would like to thank Kristin Rosowski, Birgit Korte, Stephanie Tautges, and Don Marvin Voss for their excellent technical assistance. This work was supported by the PROFILE project, which is cofunded by the European Union (European Regional Development Fund - Investing in your future) and the 2780



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