University of Groningen
Role of hepatic glucose signaling in the development of liver disease
Lei, Yu
DOI:
10.33612/diss.126530476
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Publication date: 2020
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Lei, Y. (2020). Role of hepatic glucose signaling in the development of liver disease. University of Groningen. https://doi.org/10.33612/diss.126530476
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92 Table S 7. S YB R Gr ee n pri m ers use d fo r Ch IP -q PCR . Regi on Fo rw ar d pri m er 5’ -3 ’ Re ve rs e pr im er 5 ’-3 ’ L-pk GC T CT G CAG AC A GG C CAA AG TC T T GC C AA T GG A AG C CT T G Cy p8 b1 re gi on a a GA G A CG A GG A AA G AG A TG T G CA C CG A CT G CT C AC A TT C C Cy p8 b1 re gi on b a GA G C TG A AC C TG A AC A GT A G CA G AG G CT C GG A CG T G Cy p8 b1 re gi on c a AC C ACG T CC GA G CCT CT G GG A A TT G CT TT A TG T G GC Cy p8 b1 re gi on d a GG T GGG CT C AA G GC A G GC T G AC TA G AG A G AC G AT G Cy p8 b1 re gi on -2300 CT G C AG G AC A GA T TT C AT CT T G TC A AC T G CA G AA T GT G TT A GG A C Cy p8 b1 re gi on -1500 AG G C CC C AC A GA TA G A TT C A CT G A GC A TC T GT C AG G GT GA Cy p8 b1 re gi on -280 TA A G GA G AC A CC G TC T CT A C GA G A CC T GA C AT C CC T CT A C Cy p8 b1 re gi on -100 TT G C AG A GG A CG A TA C C AAA GT G CG T GTC TG T G Cy p8 b1 re gi on 1 CA G CG C T GT A GA G CT GA C A A CA C TG T ACA C CA CA G CG T CA Cy p8 b1 re gi on 500 TC C T GA G CT TA T T CG G CT AC A CG G AA C TT C CT G AA C AG C TC Cy p8 b1 re gi on 1000 CAG C GG AC A AG A GT A CC A GA GG G G TC C AT G TG TA C T GA G AG Cy p8 b1 re gi on 2000 CG A TG C C CT TA C TC C A AA T C CT C GA T TCC AT T GA G CA A CA Cy p8 b1 re gi on 5000 TG G AA G CTG C TG A GA A AG TG CTC A GG TC C TG G CT T TTG TC a Re gi on s a re e xp la in ed in th e m an us cr ip t 93
Chapter
ChREBP promotes hepatocyte
proliferation in vitro but protects against
oncogenic hepatocyte transformation in
a mouse model for Glycogen Storage
Disease type 1a
Yu Lei1,4, Martijn G.S. Rutten1, Vincent W. Bloks1, Mirjam H. Koster1,
Rachel Thomas3, Gilles Mithieux5,6,7,Jiang Gu4,Fabienne Rajas5,6,7,Alain de
Bruin1,3, Folkert Kuipers1,2 and Maaike H. Oosterveer1
Departments of 1Pediatrics and 2Laboratory Medicine, University of
Groningen, University Medical Center Groningen, The Netherlands. 3Dutch
Molecular Pathology Center, Faculty of Veterinary Medicine, Utrecht University, 3584 CL Utrecht, The Netherlands. 4Department of Pathology
and Provincial Key Laboratory of Infectious Diseases and Immunopathology, Collaborative and Creative Center, Molecular Diagnosis and Personalized Medicine, Shantou University Medical College, Shantou, Guangdong, China. 5Institut National de la Santé et de la Recherche
Médicale, U1213, Lyon, F-69008 6Université de Lyon, Lyon, F-69008 and 7Université Lyon 1, Villeurbanne, F-69622, France
In preparation
9393
Chapter
ChREBP promotes hepatocyte
proliferation in vitro but protects against
oncogenic hepatocyte transformation in
a mouse model for Glycogen Storage
Disease type 1a
Yu Lei1,4, Martijn G.S. Rutten1, Vincent W. Bloks1, Mirjam H. Koster1,
Rachel Thomas3, Gilles Mithieux5,6,7,Jiang Gu4,Fabienne Rajas5,6,7,Alain de
Bruin1,3, Folkert Kuipers1,2 and Maaike H. Oosterveer1
Departments of 1Pediatrics and 2Laboratory Medicine, University of
Groningen, University Medical Center Groningen, The Netherlands. 3Dutch
Molecular Pathology Center, Faculty of Veterinary Medicine, Utrecht University, 3584 CL Utrecht, The Netherlands. 4Department of Pathology
and Provincial Key Laboratory of Infectious Diseases and Immunopathology, Collaborative and Creative Center, Molecular Diagnosis and Personalized Medicine, Shantou University Medical College, Shantou, Guangdong, China. 5Institut National de la Santé et de la Recherche
Médicale, U1213, Lyon, F-69008 6Université de Lyon, Lyon, F-69008 and 7Université Lyon 1, Villeurbanne, F-69622, France
94 Abstract
Glycogen Storage Disease type 1a (GSD Ia) is a rare inherited metabolic disease caused by defective glucose-6-phosphatase enzyme activity. A major long-term complication of this disorder is the development of liver tumors, but the underlying molecular mechanisms are unresolved. Carbohydrate Response Element Binding Protein (ChREBP) is a glucose-sensitive transcription factor that supports cellular proliferation in vitro, while loss of hepatic ChREBP expression protects against liver tumor development in mice. Although ChREBP is known to be constitutively active in GSD I, its contribution to liver tumor susceptibility in GSD Ia has as yet not been addressed. In the current study we assessed the oncogenic role of ChREBP in cultured immortalized human hepatocytes (IHHs) and hepatocyte-specific G6pc knockout (L-G6pc-/-) mice, a preclinical model for hepatic GSD Ia. Our data indicate that lowering ChREBP expression induces p53-activation in both IHHs and L-G6pc -/-hepatocytes. Interestingly, ChREBP knockdown in IHHs induced cell cycle inhibition and arrested proliferation, while it induced hepatocyte death, mitosis, DNA damage and chromosomal instability in L-G6pc-/- mice. Our findings therefore show that inhibition of ChREBP activity differentially affects hepatocyte fate in vitro and in vivo, and indicate a context-dependent oncogenic role of ChREBP in hepatocytes.
95 Introduction
Carbohydrate Response Element Binding Protein (ChREBP) is a glucose-sensitive transcription factor (1) and a key mediator of cellular glucose signaling in organs that control and integrate systemic glucose and lipid metabolism, such as liver, adipose tissue and pancreas. ChREBP regulates the transcription of genes that encode enzymes involved in glycolysis, de novo lipogenesis and the pentose phosphate pathway (2). Activation of these three metabolic pathways represents a typical hallmark of many cancer cells (3) and this metabolic shift serves to support proliferation and growth through macromolecule synthesis, ATP production, and enhanced oxidative defense (4). In recent years, it has become clear that increased ChREBP expression is linked to the incidence and prognosis of several types of cancer, including hepatocellular carcinoma (HCC) (5-10). On the other hand, reducing ChREBP expression in vitro inhibits cell proliferation through oxidative stress-induced, p53-mediated cell cycle arrest (11). These findings indicate that ChREBP serves as a competence factor for cell growth. Preclinical in vivo studies furthermore showed that hepatocytes from ChREBP knockout mice show lower proliferation rates during liver repopulation (12) and that full-body ChREBP knockout mice are protected against HCC development in an oncogene-specific manner (8, 12).
Glycogen Storage Disease type 1 (GSD I) is a rare inborn error of metabolism caused by mutations in the glucose-6-phosphatase enzyme (G6PC; GSD type Ia) or the glucose-6-phosphate transporter (SLC37A4; GSD type Ib). GSD Ia/b patients display a variety of biochemical symptoms, including fasting hypoglycemia, hepatomegaly, hyperlipidemia, hyperlactatemia and hyperuricemia. One of the major long-term complications of GSD Ia/b is the development of hepatocellular adenomas (HCAs) that may progress into HCCs, affecting more than two-thirds of the patients in young adulthood (13). Although previous research has suggested that several deranged signaling pathways may contribute to liver tumor progression in mouse models for GSD Ia (14-16), the molecular mechanisms that drive tumor initiation remain as yet largely unresolved. In GSD Ia/b liver, ChREBP activity, glycolysis, de novo lipogenesis, and pentose phosphate pathway are all enhanced (13, 17-21) (Chapter 2). However, the consequence of constitutively activated ChREBP on hepatocyte proliferation and liver tumor susceptibility in GSD Ia has not yet been established.
In the current study we therefore investigated the role of ChREBP on hepatocyte proliferation in immortalized human hepatocytes (IHHs) as well as hepatocyte-specific G6pc knockout (L-G6pc-/-) mice, a preclinical model for hepatic GSD Ia (22), at an early, pretumoral disease stage. Our data indicate that experimental suppression of ChREBP expression induces p53-activation in both IHHs and hepatocytes from L-G6pc-/- mice. Importantly, reduced ChREBP expression arrested proliferation of IHHs, while it induced hepatocyte death, mitosis, DNA damage and chromosomal instability in L-G6pc-/- mice. These insights are of importance for clinical translation of potential ChREBP-targeting interventions.
94 Abstract
Glycogen Storage Disease type 1a (GSD Ia) is a rare inherited metabolic disease caused by defective glucose-6-phosphatase enzyme activity. A major long-term complication of this disorder is the development of liver tumors, but the underlying molecular mechanisms are unresolved. Carbohydrate Response Element Binding Protein (ChREBP) is a glucose-sensitive transcription factor that supports cellular proliferation in vitro, while loss of hepatic ChREBP expression protects against liver tumor development in mice. Although ChREBP is known to be constitutively active in GSD I, its contribution to liver tumor susceptibility in GSD Ia has as yet not been addressed. In the current study we assessed the oncogenic role of ChREBP in cultured immortalized human hepatocytes (IHHs) and hepatocyte-specific G6pc knockout (L-G6pc-/-) mice, a preclinical model for hepatic GSD Ia. Our data indicate that lowering ChREBP expression induces p53-activation in both IHHs and L-G6pc -/-hepatocytes. Interestingly, ChREBP knockdown in IHHs induced cell cycle inhibition and arrested proliferation, while it induced hepatocyte death, mitosis, DNA damage and chromosomal instability in L-G6pc-/- mice. Our findings therefore show that inhibition of ChREBP activity differentially affects hepatocyte fate in vitro and in vivo, and indicate a context-dependent oncogenic role of ChREBP in hepatocytes.
95 Introduction
Carbohydrate Response Element Binding Protein (ChREBP) is a glucose-sensitive transcription factor (1) and a key mediator of cellular glucose signaling in organs that control and integrate systemic glucose and lipid metabolism, such as liver, adipose tissue and pancreas. ChREBP regulates the transcription of genes that encode enzymes involved in glycolysis, de novo lipogenesis and the pentose phosphate pathway (2). Activation of these three metabolic pathways represents a typical hallmark of many cancer cells (3) and this metabolic shift serves to support proliferation and growth through macromolecule synthesis, ATP production, and enhanced oxidative defense (4). In recent years, it has become clear that increased ChREBP expression is linked to the incidence and prognosis of several types of cancer, including hepatocellular carcinoma (HCC) (5-10). On the other hand, reducing ChREBP expression in vitro inhibits cell proliferation through oxidative stress-induced, p53-mediated cell cycle arrest (11). These findings indicate that ChREBP serves as a competence factor for cell growth. Preclinical in vivo studies furthermore showed that hepatocytes from ChREBP knockout mice show lower proliferation rates during liver repopulation (12) and that full-body ChREBP knockout mice are protected against HCC development in an oncogene-specific manner (8, 12).
Glycogen Storage Disease type 1 (GSD I) is a rare inborn error of metabolism caused by mutations in the glucose-6-phosphatase enzyme (G6PC; GSD type Ia) or the glucose-6-phosphate transporter (SLC37A4; GSD type Ib). GSD Ia/b patients display a variety of biochemical symptoms, including fasting hypoglycemia, hepatomegaly, hyperlipidemia, hyperlactatemia and hyperuricemia. One of the major long-term complications of GSD Ia/b is the development of hepatocellular adenomas (HCAs) that may progress into HCCs, affecting more than two-thirds of the patients in young adulthood (13). Although previous research has suggested that several deranged signaling pathways may contribute to liver tumor progression in mouse models for GSD Ia (14-16), the molecular mechanisms that drive tumor initiation remain as yet largely unresolved. In GSD Ia/b liver, ChREBP activity, glycolysis, de novo lipogenesis, and pentose phosphate pathway are all enhanced (13, 17-21) (Chapter 2). However, the consequence of constitutively activated ChREBP on hepatocyte proliferation and liver tumor susceptibility in GSD Ia has not yet been established.
In the current study we therefore investigated the role of ChREBP on hepatocyte proliferation in immortalized human hepatocytes (IHHs) as well as hepatocyte-specific G6pc knockout (L-G6pc-/-) mice, a preclinical model for hepatic GSD Ia (22), at an early, pretumoral disease stage. Our data indicate that experimental suppression of ChREBP expression induces p53-activation in both IHHs and hepatocytes from L-G6pc-/- mice. Importantly, reduced ChREBP expression arrested proliferation of IHHs, while it induced hepatocyte death, mitosis, DNA damage and chromosomal instability in L-G6pc-/- mice. These insights are of importance for clinical translation of potential ChREBP-targeting interventions.
95
96 Materials and Methods
Cell culture and siRNA transfection
Immortalized human hepatocytes (IHHs) (23) were cultured in 0.1% porcine-derived gelatin-coated (Sigma G1890) flasks with complete Williams’ medium E containing 2 mM glutamine and supplemented with 10% fetal calf serum FCS, 1% Penicillin/Streptomycin, 20 mU/mL bovine insulin (Sigma-Aldrich), 50 nmol/L dexamethasone (Sigma-Aldrich) (normal growth medium) at 37 °C in a humidified 5% CO2 atmosphere. pCMVS4/ChREBPα and pCMVS4/ChREBPβ (kind gifts from M. Herman) were shuttled to pcDNA3.1 using cloning PCR. IHH cells grown to 70% confluency were transfected with 2.5 μg pcDNA3.1/ChREBPα, pcDNA3.1/ChREBPβ or pcDNA3.1 control plasmid for 48 hours using Lipofectamine 3000 Transfection Reagent (Invitrogen) according to the manufacturer’s instruction. For siRNA transfection, cells were plated in 12-well plates at a density of 2 x 105 cells per well.
Non-specific control small interfering RNAs (siRNAs) and oligonucleotides targeting ChREBP were commercially synthesized (GenePharma, China). The siChREBP
sequences were 5ʹ-GCACCCUUGGCAAACCUUUUU-3ʹ and
5ʹ-AAAGGUUUGCCAAGGGUGCUU-3ʹ. Control scrambled siRNA sequences were 5ʹ-UUCUCCGAACGUGUCACGUUU-3ʹ and 5ʹ-ACGUGACACGUUCGGAGAAUU-3ʹ. For cell apoptosis and cell cycle assessments, IHH cells were transfected for 48 hours using Lipofectamine™ RNAiMAX (Invitrogen) according to the manufacturer’s protocol with 10 pmol ChREBP/control siRNA per well in Williams E medium containing 2 mM glutamine and supplemented with 2% FCS, 20 mU/mL insulin and 50 nM dexamethasone, after which they were used for cell apoptosis and cell cycle analyses.
Cell proliferation assays
IHH cells were plated at 5 x 104 cells per well in 12-well plates and cultured with
normal growth medium for 24 hours. At 48, 96, and 144 hours after transfection with either control or ChREBP siRNAs, cells were fixed using 1% glutaraldehyde for 15 minutes and stored in PBS at 4 degree until further analysis. 0.1% crystal violate (Solarbio, China) was used to stain cells for 30 minutes after which the dye was extracted from the cells using 10% acetic acid. The absorbance of extracts was measured at 490 nm using an ELISA plate reader (Bio-Rad, USA). Quadruple wells were used for each experimental condition, and all the experiments were repeated at least three times.
Cell apoptosis assays
IHH cells were seeded at 1× 105 per well in 12-well plates and cultured with normal
growth medium for 24 hours. On the second day, IHH cells were transfected with ChREBP or control siRNAs. After 48 hours, IHH cells were harvested with trypsin and washed 2 times in ice-cold PBS, resuspended in 100 μL of Binding Buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) and incubated with Annexin
V-FITC (4A Biotech, China) for 5 minutes at 4 °C in the dark. After staining, the cells were incubated with propidium iodide (PI) for 5 minutes at 4°C in the dark and subsequently assayed by flow cytometry (Beckman Coulter, Germany) (24). FlowJo software (Becton, Dickinson & Company, USA) was used to analyze the fractions of
97
cells in early (only Annexin V positivity) or late (combined Annexin V and PI positivity) apoptosis.
Cell cycle analysis
Cell cycle analysis was performed using a cell cycle kit (Shanghai BestBio, China) according to the manufacturer’s instructions (25). Briefly, IHH cells were seeded at 1× 105 per well in 12-well plates and cultured in normal growth medium for 24 hours.
On the second day, IHH cells were transfected with ChREBP or control siRNAs. After 48 hours, IHH cells were lysed using trypsin and washed 2 times in ice-cold PBS and fixed with 70% ethanol at -20°C for 1 hour. Samples were washed 2 times again with cold PBS and incubated with 20 μL RNase A at 37°C for 30 minutes, followed by incubation with 400 μL PI for 30 minutes at 4°C in the dark, and finally assayed by flow cytometry (Beckman Coulter, Germany). FlowJo software (Becton, Dickinson & Company, USA) was used to analyze cell cycle stage based on PI incorporation. Construction, production and in vivo transduction of shRNAs using
self-complementary AAV vectors
To construct the self-complementary AAV (scAAV) 2/8-U6-ChREBP, the scAAV2-LP1-hFIXco backbone vector (26) was restricted with BamHI and BbsI and the 3493 bp fragment was isolated and ligated. Restriction with BamHI removed hFIXco and partially deleted the LP1 promoter. The U6 promoter driving the expression of the construct was cloned into the vector in antisense orientation. shRNA construct directed against ChREBP (shChREBP) and scramble (shSCR) construct were ordered as oligonucleotides (shChREBP; 5’-aat tcA AAA AAT GTA GTT TGA AGA TGT GGG TCT CGA GAC CCA CAT CTT CAA ACT ACA TC-3’ and 3’-ggc caG ATG TAG TTT GAA GAT GTG GGT CTC GAG ACC CAC ATC TTC AAA CTA CAT TTT TT-5’, shSCR; 5’-aat tcG TTG TAA GTG GAG GTT TAA GTC TCG AGA CTT AAA CCT CCA CTT ACA ACA CCG GT-3’ and 3’-ggc caA CCG GTG TTG TAA GTG GAG GTT TAA GTC TCG AGA CTT AAA CCT CCA CTT ACA AC-5’), denatured at 99 °C for 10 minutes and annealed by cooling down to RT. The duplex oligonucleotides were cloned into the vector using EcoRI and AgeI. Production, purification and titration of the AAV2/8 viruses encoding the shRNA directed against ChREBP (AAV-shChREBP) and the scrambled control (AAV-shSCR) were performed as described (27, 28).
Mouse experimentation
Male adult (13-18 weeks) B6.G6pclox/lox and B6.G6pclox/lox.SAcreERT2/w mice (22) were
housed in a light- and temperature-controlled facility (lights on 7AM-7PM) and fed a standard laboratory chow diet (RMH-B, Abdiets, Woerden). They were injected with AAV-shChREBP or AAV-shSCR viruses (1*1012 virus particles in 100 μL PBS/mouse) via
the retro-orbital plexus under isoflurane anesthesia. To induce hepatocyte-specific excision of G6pc exon 3, twelve days after AAV-shRNA administration, all mice received i.p. injections of tamoxifen for 5 consecutive days as described (22), hence generating hepatocyte-specific G6pc-deficient mice (L-G6pc-/-) and wildtype
littermates (L-G6pc+/+). Animals were sacrificed by cardiac puncture under isoflurane
anesthesia 10 days after the final tamoxifen injection and tissues were rapidly excised and stored. Experimental procedures were approved by the Ethics Committees for Animal Experiments of the University of Groningen.
96 Materials and Methods
Cell culture and siRNA transfection
Immortalized human hepatocytes (IHHs) (23) were cultured in 0.1% porcine-derived gelatin-coated (Sigma G1890) flasks with complete Williams’ medium E containing 2 mM glutamine and supplemented with 10% fetal calf serum FCS, 1% Penicillin/Streptomycin, 20 mU/mL bovine insulin (Sigma-Aldrich), 50 nmol/L dexamethasone (Sigma-Aldrich) (normal growth medium) at 37 °C in a humidified 5% CO2 atmosphere. pCMVS4/ChREBPα and pCMVS4/ChREBPβ (kind gifts from M. Herman) were shuttled to pcDNA3.1 using cloning PCR. IHH cells grown to 70% confluency were transfected with 2.5 μg pcDNA3.1/ChREBPα, pcDNA3.1/ChREBPβ or pcDNA3.1 control plasmid for 48 hours using Lipofectamine 3000 Transfection Reagent (Invitrogen) according to the manufacturer’s instruction. For siRNA transfection, cells were plated in 12-well plates at a density of 2 x 105 cells per well.
Non-specific control small interfering RNAs (siRNAs) and oligonucleotides targeting ChREBP were commercially synthesized (GenePharma, China). The siChREBP
sequences were 5ʹ-GCACCCUUGGCAAACCUUUUU-3ʹ and
5ʹ-AAAGGUUUGCCAAGGGUGCUU-3ʹ. Control scrambled siRNA sequences were 5ʹ-UUCUCCGAACGUGUCACGUUU-3ʹ and 5ʹ-ACGUGACACGUUCGGAGAAUU-3ʹ. For cell apoptosis and cell cycle assessments, IHH cells were transfected for 48 hours using Lipofectamine™ RNAiMAX (Invitrogen) according to the manufacturer’s protocol with 10 pmol ChREBP/control siRNA per well in Williams E medium containing 2 mM glutamine and supplemented with 2% FCS, 20 mU/mL insulin and 50 nM dexamethasone, after which they were used for cell apoptosis and cell cycle analyses.
Cell proliferation assays
IHH cells were plated at 5 x 104 cells per well in 12-well plates and cultured with
normal growth medium for 24 hours. At 48, 96, and 144 hours after transfection with either control or ChREBP siRNAs, cells were fixed using 1% glutaraldehyde for 15 minutes and stored in PBS at 4 degree until further analysis. 0.1% crystal violate (Solarbio, China) was used to stain cells for 30 minutes after which the dye was extracted from the cells using 10% acetic acid. The absorbance of extracts was measured at 490 nm using an ELISA plate reader (Bio-Rad, USA). Quadruple wells were used for each experimental condition, and all the experiments were repeated at least three times.
Cell apoptosis assays
IHH cells were seeded at 1× 105 per well in 12-well plates and cultured with normal
growth medium for 24 hours. On the second day, IHH cells were transfected with ChREBP or control siRNAs. After 48 hours, IHH cells were harvested with trypsin and washed 2 times in ice-cold PBS, resuspended in 100 μL of Binding Buffer (10 mM Hepes/NaOH, pH 7.4, 140 mM NaCl, 2.5 mM CaCl2) and incubated with Annexin
V-FITC (4A Biotech, China) for 5 minutes at 4 °C in the dark. After staining, the cells were incubated with propidium iodide (PI) for 5 minutes at 4°C in the dark and subsequently assayed by flow cytometry (Beckman Coulter, Germany) (24). FlowJo software (Becton, Dickinson & Company, USA) was used to analyze the fractions of
97
cells in early (only Annexin V positivity) or late (combined Annexin V and PI positivity) apoptosis.
Cell cycle analysis
Cell cycle analysis was performed using a cell cycle kit (Shanghai BestBio, China) according to the manufacturer’s instructions (25). Briefly, IHH cells were seeded at 1× 105 per well in 12-well plates and cultured in normal growth medium for 24 hours.
On the second day, IHH cells were transfected with ChREBP or control siRNAs. After 48 hours, IHH cells were lysed using trypsin and washed 2 times in ice-cold PBS and fixed with 70% ethanol at -20°C for 1 hour. Samples were washed 2 times again with cold PBS and incubated with 20 μL RNase A at 37°C for 30 minutes, followed by incubation with 400 μL PI for 30 minutes at 4°C in the dark, and finally assayed by flow cytometry (Beckman Coulter, Germany). FlowJo software (Becton, Dickinson & Company, USA) was used to analyze cell cycle stage based on PI incorporation. Construction, production and in vivo transduction of shRNAs using
self-complementary AAV vectors
To construct the self-complementary AAV (scAAV) 2/8-U6-ChREBP, the scAAV2-LP1-hFIXco backbone vector (26) was restricted with BamHI and BbsI and the 3493 bp fragment was isolated and ligated. Restriction with BamHI removed hFIXco and partially deleted the LP1 promoter. The U6 promoter driving the expression of the construct was cloned into the vector in antisense orientation. shRNA construct directed against ChREBP (shChREBP) and scramble (shSCR) construct were ordered as oligonucleotides (shChREBP; 5’-aat tcA AAA AAT GTA GTT TGA AGA TGT GGG TCT CGA GAC CCA CAT CTT CAA ACT ACA TC-3’ and 3’-ggc caG ATG TAG TTT GAA GAT GTG GGT CTC GAG ACC CAC ATC TTC AAA CTA CAT TTT TT-5’, shSCR; 5’-aat tcG TTG TAA GTG GAG GTT TAA GTC TCG AGA CTT AAA CCT CCA CTT ACA ACA CCG GT-3’ and 3’-ggc caA CCG GTG TTG TAA GTG GAG GTT TAA GTC TCG AGA CTT AAA CCT CCA CTT ACA AC-5’), denatured at 99 °C for 10 minutes and annealed by cooling down to RT. The duplex oligonucleotides were cloned into the vector using EcoRI and AgeI. Production, purification and titration of the AAV2/8 viruses encoding the shRNA directed against ChREBP (AAV-shChREBP) and the scrambled control (AAV-shSCR) were performed as described (27, 28).
Mouse experimentation
Male adult (13-18 weeks) B6.G6pclox/lox and B6.G6pclox/lox.SAcreERT2/w mice (22) were
housed in a light- and temperature-controlled facility (lights on 7AM-7PM) and fed a standard laboratory chow diet (RMH-B, Abdiets, Woerden). They were injected with AAV-shChREBP or AAV-shSCR viruses (1*1012 virus particles in 100 μL PBS/mouse) via
the retro-orbital plexus under isoflurane anesthesia. To induce hepatocyte-specific excision of G6pc exon 3, twelve days after AAV-shRNA administration, all mice received i.p. injections of tamoxifen for 5 consecutive days as described (22), hence generating hepatocyte-specific G6pc-deficient mice (L-G6pc-/-) and wildtype
littermates (L-G6pc+/+). Animals were sacrificed by cardiac puncture under isoflurane
anesthesia 10 days after the final tamoxifen injection and tissues were rapidly excised and stored. Experimental procedures were approved by the Ethics Committees for Animal Experiments of the University of Groningen.
97
98 Real-time PCR
For both mouse liver tissue and IHH samples, total RNA was isolated by TRI-Reagent (Sigma-Aldrich, USA) according to the manufacturer’s protocol. cDNA was obtained by reverse transcription (Life Technologies) using 1 μg of total RNA. qPCR reactions were performed using the TaqMan Universal PCR Master Mix Kit or SYBR Green PCR Master Mix Kit (Takara, Japan) and an ABI 7500 PCR system (Applied Biosystems). For liver tissue, mRNA levels were calculated based on a pooled calibration curve, expressed relative to the expression of Ppig (cyclophilin). For IHH cells, data were quantified according to the 2-ΔΔCq method, normalized to β2 microglobulin (B2M),
and normalized to the average values in the siControl-treated group. The sequences of the primers and probes are listed in Table S1.
RNA-sequencing and gene set enrichment analysis (GSEA)
For RNA sequencing, initial quality check and RNA quantification of the samples was performed by capillary electrophoresis using the LabChip GX (Perkin Elmer). Non-degraded RNA-samples were used for subsequent sequencing analysis. Sequence libraries were generated using the 3’QuantSeq sample preparation kits (LeXogen). The obtained cDNA fragment libraries were sequenced on an Illumina HiSeq2500 using default parameters (single read 1x50bp) in pools of multiple samples. The fastQ files where aligned to build Mus_musculus GRCm38 Ensemble Release 82 reference genome using HISAT (29) with default settings. Before gene quantification SAMtools was used to sort the aligned reads (30). The gene level quantification was performed by HTSeq-count (31). The extracted raw count file (ENSG, counts) was analyzed in the MADMAX (32). After transformation (log2CPM),
normalization (Voom) (33) was performed to generate the differential expressed genes. Exploration of the microarray data was performed using gene set enrichment analysis (GSEA) software version 2.2.4. The versions of the gene set database used were Mm_PathwaysOnly_GS.v3.gmt and h.all.v6.1.symbols.gm with addition of a custom for chromosomal instability (CIN) gene set (CIN29; Table S2).
SDS-PAGE and Western blot
To analyze cytosolic protein expression, IHH cells were washed twice with ice-cold PBS and lysed in RIPA lysis buffer (Millipore, USA) for 30 minutes on ice. Lysates were clarified by centrifugation at 12,000 xg for 15 minutes at 4°C. The protein concentrations in the cell lysates were measured using a BCA kit (Pierce Biotechnology, USA). Identical amounts of lysate proteins (30 μg/well) were separated by 10% SDS-polyacrylamide gel electrophoresis. Proteins were transferred onto a polyvinylidene difluoride membrane and incubated in a blocking solution consisting of 5% powered milk in TBST (10 mmol/L Tris–HCl (pH 8.0), 150 mmol/L NaCl, and 0.1% Tween 20) for 1 h, followed by immunoblotting with the respective antibodies. Primary antibodies were incubated with rabbit anti-mouse ChREBP (Novus, NB400-135), rabbit anti-mouse β-actin (Cell signaling, 4967S), rabbit anti-human p21 (Abcam, EFR362), rabbit anti-human phospho-p53 (Cell signaling, 9284) and anti-human Caspase 3 (Cell signaling, 9662) at 4°C overnight. Subsequently the membranes were washed with TBST for 30 minutes at room temperature before incubation with secondary antibodies for 2 hours at room temperature. Goat anti-rabbit IgG-HRP (Dako, Carpinteria, CA) and rabbit anti-goat
99
IgG-HRP (Dako, Carpinteria, CA) were used as secondary antibodies. After incubation of secondary antibodies, membranes were washed with TBST for 30 minutes at room temperature. Immunoreactive proteins were detected with the Odyssey Infrared imager according to the manufacturer’s protocol (Li-Cor Biosciences, NE). Western Blot band intensities were quantified using Image J software (National Institutes of Health, Bethesda, MD).
Oxidized and reduced glutathione analysis
Freeze-dried liver (2 mg) was collected in a 2 mL tube followed by the addition of 1 mL ice-cold methanol:water (1:1) containing internal standards. For the extraction of metabolites, 1 mL of chloroform was added and the samples were needle sonicated (8 watt, 40 joule). The homogenate was centrifuged for 5 minutes at 14.000 rpm at 4°C. The “polar” top layer was transferred to a new 1.5 mL tube and dried in a vacuum concentrator. Dried samples were dissolved in 100 µL methanol/water (6/4; v/v). The metabolomic analysis was performed as described previously (34, 35) and interpretation of the data was performed in the Xcalibur software (Thermo scientific). Metabolite levels were normalized to the average values in the L-G6pc+/+/shSCR-treated group.
Histological analysis
Mouse livers were trimmed, fixed in 4% formalin solution, embedded in paraffin, blocked, sectioned (4 μm), and stained with hematoxylin and eosin (H&E).Stained sections were examined histologically for hepatocellular vacuolization, inflammatory foci, single cell death and mitotic figures by a veterinary pathologist. Immunohistochemistry was carried out following an established protocol (36). Briefly, paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated in a graded series of alcohol. Antigen retrieval was performed using 10mM citrate buffer pH 6.0 in a microwave (LG Intellowave, 15 minutes at 640 Watt) and the slides were subsequently left to cool to room temperature for 30 minutes. Endogenous peroxidase activity was blocked with 1% H2O2 in methanol for 30 minutes. The unspecific antigens were blocked in 10% normal goat serum for 30 minutes. Sections were incubated with primary antibodies rabbit anti Ki67 (RM-9106, Thermo, at 1:50 dilution), and pH3 (06-570,Millipore,at 1:400 dilution), γH2AX (9718S, Millipore, at 1:500 dilution) and c-C3 (AF835, R&D systems, at 1:400 dilution) at 4°C overnight and subsequently incubated with biotinylated goat-anti-rabbit secondary antibody (Vector Labs BA-1000 at 1:250) for 30 minutes at room temperature. After 30 minutes incubation with Avidin-biotin ABC complex (PK-4000, Vector), the sections were washed and colorized with a 3,3ʹ-diaminobenzidine (DAB) (Sigma, D5637) for 10 minutes and subsequently counterstained with hematoxylin. Slides were scanned using a Nikon E800 microscope. Positive hepatocytes were counted by examination of at least three random high-power microscopic fields (magnification: 200x or 400x) in each tissue section by a veterinary pathologist. As significant differences in hepatocyte vacuolation were observed between groups (Chapter 2), scores for Ki67-, pH3-, γH2AX-, and cC3-positivity, as well as the incidence of mitotic figures, single cell death and inflammatory foci were corrected for the average hepatocyte number per microscopic field. In order to do this, average hepatocyte sizes for each individual were quantified on H&E stained sections using Image J. Corrected scores for Ki67, 98
98 Real-time PCR
For both mouse liver tissue and IHH samples, total RNA was isolated by TRI-Reagent (Sigma-Aldrich, USA) according to the manufacturer’s protocol. cDNA was obtained by reverse transcription (Life Technologies) using 1 μg of total RNA. qPCR reactions were performed using the TaqMan Universal PCR Master Mix Kit or SYBR Green PCR Master Mix Kit (Takara, Japan) and an ABI 7500 PCR system (Applied Biosystems). For liver tissue, mRNA levels were calculated based on a pooled calibration curve, expressed relative to the expression of Ppig (cyclophilin). For IHH cells, data were quantified according to the 2-ΔΔCq method, normalized to β2 microglobulin (B2M),
and normalized to the average values in the siControl-treated group. The sequences of the primers and probes are listed in Table S1.
RNA-sequencing and gene set enrichment analysis (GSEA)
For RNA sequencing, initial quality check and RNA quantification of the samples was performed by capillary electrophoresis using the LabChip GX (Perkin Elmer). Non-degraded RNA-samples were used for subsequent sequencing analysis. Sequence libraries were generated using the 3’QuantSeq sample preparation kits (LeXogen). The obtained cDNA fragment libraries were sequenced on an Illumina HiSeq2500 using default parameters (single read 1x50bp) in pools of multiple samples. The fastQ files where aligned to build Mus_musculus GRCm38 Ensemble Release 82 reference genome using HISAT (29) with default settings. Before gene quantification SAMtools was used to sort the aligned reads (30). The gene level quantification was performed by HTSeq-count (31). The extracted raw count file (ENSG, counts) was analyzed in the MADMAX (32). After transformation (log2CPM),
normalization (Voom) (33) was performed to generate the differential expressed genes. Exploration of the microarray data was performed using gene set enrichment analysis (GSEA) software version 2.2.4. The versions of the gene set database used were Mm_PathwaysOnly_GS.v3.gmt and h.all.v6.1.symbols.gm with addition of a custom for chromosomal instability (CIN) gene set (CIN29; Table S2).
SDS-PAGE and Western blot
To analyze cytosolic protein expression, IHH cells were washed twice with ice-cold PBS and lysed in RIPA lysis buffer (Millipore, USA) for 30 minutes on ice. Lysates were clarified by centrifugation at 12,000 xg for 15 minutes at 4°C. The protein concentrations in the cell lysates were measured using a BCA kit (Pierce Biotechnology, USA). Identical amounts of lysate proteins (30 μg/well) were separated by 10% SDS-polyacrylamide gel electrophoresis. Proteins were transferred onto a polyvinylidene difluoride membrane and incubated in a blocking solution consisting of 5% powered milk in TBST (10 mmol/L Tris–HCl (pH 8.0), 150 mmol/L NaCl, and 0.1% Tween 20) for 1 h, followed by immunoblotting with the respective antibodies. Primary antibodies were incubated with rabbit anti-mouse ChREBP (Novus, NB400-135), rabbit anti-mouse β-actin (Cell signaling, 4967S), rabbit anti-human p21 (Abcam, EFR362), rabbit anti-human phospho-p53 (Cell signaling, 9284) and anti-human Caspase 3 (Cell signaling, 9662) at 4°C overnight. Subsequently the membranes were washed with TBST for 30 minutes at room temperature before incubation with secondary antibodies for 2 hours at room temperature. Goat anti-rabbit IgG-HRP (Dako, Carpinteria, CA) and rabbit anti-goat
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IgG-HRP (Dako, Carpinteria, CA) were used as secondary antibodies. After incubation of secondary antibodies, membranes were washed with TBST for 30 minutes at room temperature. Immunoreactive proteins were detected with the Odyssey Infrared imager according to the manufacturer’s protocol (Li-Cor Biosciences, NE). Western Blot band intensities were quantified using Image J software (National Institutes of Health, Bethesda, MD).
Oxidized and reduced glutathione analysis
Freeze-dried liver (2 mg) was collected in a 2 mL tube followed by the addition of 1 mL ice-cold methanol:water (1:1) containing internal standards. For the extraction of metabolites, 1 mL of chloroform was added and the samples were needle sonicated (8 watt, 40 joule). The homogenate was centrifuged for 5 minutes at 14.000 rpm at 4°C. The “polar” top layer was transferred to a new 1.5 mL tube and dried in a vacuum concentrator. Dried samples were dissolved in 100 µL methanol/water (6/4; v/v). The metabolomic analysis was performed as described previously (34, 35) and interpretation of the data was performed in the Xcalibur software (Thermo scientific). Metabolite levels were normalized to the average values in the L-G6pc+/+/shSCR-treated group.
Histological analysis
Mouse livers were trimmed, fixed in 4% formalin solution, embedded in paraffin, blocked, sectioned (4 μm), and stained with hematoxylin and eosin (H&E).Stained sections were examined histologically for hepatocellular vacuolization, inflammatory foci, single cell death and mitotic figures by a veterinary pathologist. Immunohistochemistry was carried out following an established protocol (36). Briefly, paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated in a graded series of alcohol. Antigen retrieval was performed using 10mM citrate buffer pH 6.0 in a microwave (LG Intellowave, 15 minutes at 640 Watt) and the slides were subsequently left to cool to room temperature for 30 minutes. Endogenous peroxidase activity was blocked with 1% H2O2 in methanol for 30 minutes. The unspecific antigens were blocked in 10% normal goat serum for 30 minutes. Sections were incubated with primary antibodies rabbit anti Ki67 (RM-9106, Thermo, at 1:50 dilution), and pH3 (06-570,Millipore,at 1:400 dilution), γH2AX (9718S, Millipore, at 1:500 dilution) and c-C3 (AF835, R&D systems, at 1:400 dilution) at 4°C overnight and subsequently incubated with biotinylated goat-anti-rabbit secondary antibody (Vector Labs BA-1000 at 1:250) for 30 minutes at room temperature. After 30 minutes incubation with Avidin-biotin ABC complex (PK-4000, Vector), the sections were washed and colorized with a 3,3ʹ-diaminobenzidine (DAB) (Sigma, D5637) for 10 minutes and subsequently counterstained with hematoxylin. Slides were scanned using a Nikon E800 microscope. Positive hepatocytes were counted by examination of at least three random high-power microscopic fields (magnification: 200x or 400x) in each tissue section by a veterinary pathologist. As significant differences in hepatocyte vacuolation were observed between groups (Chapter 2), scores for Ki67-, pH3-, γH2AX-, and cC3-positivity, as well as the incidence of mitotic figures, single cell death and inflammatory foci were corrected for the average hepatocyte number per microscopic field. In order to do this, average hepatocyte sizes for each individual were quantified on H&E stained sections using Image J. Corrected scores for Ki67,
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pH3, γH2AX, cC3, single cell death and inflammatory foci were expressed relative to the average values in the L-G6pc+/+/shSCR-treated group.
Hepatic Caspase-3 activity analysis
Caspase 3 activity was quantified as previously described (37). Liver samples were lysed in Cell lysis buffer for Caspase 3 (25 mM HEPES, pH 7.5, 5 mM MgCl2, 5 mM
EDTA, 2 mM PMSF, 10 μg/μL pepstatine, 10 μg/μL leupeptine). The lysate protein concentration was analyzed using a BCA kit (Pierce Biotechnology, USA). 20 μg protein extracted from each sample was added to 2 µL 2.5 mM Ac-DEVD-AMC (Biomol, P-411) and incubated at 37°C for 60 minutes. Fluorescence of the reactions was analyzed at an excitation wavelength of 380 nm and an emission wavelength of 460 nm using a fluorescence microplate reader (Bio-Rad, CA). Data were normalized to the average value in the L-G6pc+/+/shSCR-treated group.
Statistical analysis
Data in figures is presented either as box and-whisker plots indicating the sample minimum, lower quartile, median, upper quartile, and sample maximum, as scatter dot plots with median values, or as average values with standard error of the mean (SEM). Differences between two or multiple groups were tested by Mann-Whitney U-test or Kruskal-Wallis H-test followed by post-hoc Conover pairwise comparisons, respectively. Statistical significance was set at (nominal) p-values <0.05 and FDR q-values <0.25.
Results
ChREBP knockdown in immortalized human hepatocytes reduces cell proliferation by inducing p53 activity and cell cycle arrest
Previous work has shown that ChREBP silencing in human hepatoma cells inhibits cell proliferation (11). To further explore the role of ChREBP in regulating liver cell proliferation, we downregulated ChREBP expression in immortalized human hepatocytes (IHHs) using RNA interference (siRNAs). Compared to non-targeting siRNA treatment, siChREBP reduced ChREBPα and -β mRNA levels by 45% and 75%, respectively, while protein levels were decreased by 50% and 34% (Fig. 1A). The mRNA expression levels of the established ChREBP target genes liver-type pyruvate kinase (PKLR), elongation of very long chain fatty acids protein 6 (ELOVL6) and stearoyl-CoA desaturase-1 (SCD1) were reduced in siChREBP- versus siControl-treated cells, while fatty acid synthase (FASN) and ATP citrate lyase (ACLY) expression remained unchanged (Fig 1B). Quantification of cell numbers showed that, at two days after ChREBP knockdown, cell proliferation was significantly inhibited in parallel to reduced antigen KI-67 (Ki67) mRNA levels as compared to siControl-treated cells (Fig. 1C). At this timepoint, the incidence of cell apoptosis was similar in siChREBP- and siControl-treated cells (Fig. 1D). However, the fraction of cells in G0-1 phase was significantly increased upon ChREBP knockdown, while the relative number of cells in S phase was reduced (Fig. 1E). The mRNA expression levels oftumor protein p53 (TP53) and its target genes cyclin-dependent kinase inhibitor 1 (CDKN1A; also known as P21), TP53-inducible glycolysis and apoptosis regulator
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pH3, γH2AX, cC3, single cell death and inflammatory foci were expressed relative to the average values in the L-G6pc+/+/shSCR-treated group.
Hepatic Caspase-3 activity analysis
Caspase 3 activity was quantified as previously described (37). Liver samples were lysed in Cell lysis buffer for Caspase 3 (25 mM HEPES, pH 7.5, 5 mM MgCl2, 5 mM EDTA, 2 mM PMSF, 10 μg/μL pepstatine, 10 μg/μL leupeptine). The lysate protein concentration was analyzed using a BCA kit (Pierce Biotechnology, USA). 20 μg protein extracted from each sample was added to 2 µL 2.5 mM Ac-DEVD-AMC (Biomol, P-411) and incubated at 37°C for 60 minutes. Fluorescence of the reactions was analyzed at an excitation wavelength of 380 nm and an emission wavelength of 460 nm using a fluorescence microplate reader (Bio-Rad, CA). Data were normalized to the average value in the L-G6pc+/+/shSCR-treated group.
Statistical analysis
Data in figures is presented either as box and-whisker plots indicating the sample minimum, lower quartile, median, upper quartile, and sample maximum, as scatter dot plots with median values, or as average values with standard error of the mean (SEM). Differences between two or multiple groups were tested by Mann-Whitney U-test or Kruskal-Wallis H-test followed by post-hoc Conover pairwise comparisons, respectively. Statistical significance was set at (nominal) p-values <0.05 and FDR q-values <0.25.
Results
ChREBP knockdown in immortalized human hepatocytes reduces cell proliferation by inducing p53 activity and cell cycle arrest
Previous work has shown that ChREBP silencing in human hepatoma cells inhibits cell proliferation (11). To further explore the role of ChREBP in regulating liver cell proliferation, we downregulated ChREBP expression in immortalized human hepatocytes (IHHs) using RNA interference (siRNAs). Compared to non-targeting siRNA treatment, siChREBP reduced ChREBPα and -β mRNA levels by 45% and 75%, respectively, while protein levels were decreased by 50% and 34% (Fig. 1A). The mRNA expression levels of the established ChREBP target genes liver-type pyruvate kinase (PKLR), elongation of very long chain fatty acids protein 6 (ELOVL6) and stearoyl-CoA desaturase-1 (SCD1) were reduced in siChREBP- versus siControl-treated cells, while fatty acid synthase (FASN) and ATP citrate lyase (ACLY) expression remained unchanged (Fig 1B). Quantification of cell numbers showed that, at two days after ChREBP knockdown, cell proliferation was significantly inhibited in parallel to reduced antigen KI-67 (Ki67) mRNA levels as compared to siControl-treated cells (Fig. 1C). At this timepoint, the incidence of cell apoptosis was similar in siChREBP- and siControl-treated cells (Fig. 1D). However, the fraction of cells in G0-1 phase was significantly increased upon ChREBP knockdown, while the relative number of cells in S phase was reduced (Fig. 1E). The mRNA expression levels of tumor protein p53 (TP53) and its target genes cyclin-dependent kinase inhibitor 1 (CDKN1A; also known as P21), TP53-inducible glycolysis and apoptosis regulator
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Figure 1. ChREBP knockdown in immortalized human hepatocytes (IHHs) reduces cell proliferation while inducing p53 activity and cell cycle arrest. A, relative mRNA and protein levels of ChREBP. Proteins were extracted from cells transfected with ChREBPα or ChREBPβ plasmids or siRNA of ChREBP or control. The bands of ChREBPα and ChREBPβ are indicated in the solid and dashed rectangles, respectively. Protein expression levels were expressed relative to β-actin. mRNA levels were expressed relative to B2M. B, relative mRNA levels of ChREBP target genes expressed relative to B2M. C, IHH cell growth curve after cells treated with siControl or siChREBP. D, cell apoptosis assessment with flow cytometry after treated with control or ChREBP siRNA for 48 hours. The right down corner window represents early apoptosis, while the right up corner window represents late apoptosis or cell death. E, cell cycle measurement with flow cytometry after treatment with control or ChREBP siRNA for 48 hours. F, relative mRNA expression of Ki67 as well as p53 target genes expressed relative to B2M. G, western blot of p21, phosphorylated-p53 and total Caspase 3. Protein expression levels were expressed relative to β-actin. Data in A, B, F and G is presented as box and-whisker plots indicating the sample minimum, lower quartile, median, upper quartile, and sample maximum. Data in C is presented as mean and SEM. n=3-4, *p<0.05, **p<0.01, ***p<0.001.
(TIGAR), E3 ubiquitin-protein ligase (MDM2) and C/EBP homology protein (CHOP) were all significantly increased in siChREBP-treated compared to control cells (Fig. 1F) and were paralleled by higher P21 and phospho-P53 protein expression levels (Fig. 1G). In agreement with unaltered numbers of Annexin V positive cells (Fig. 1D), Caspase 3 protein expression remained unaffected by ChREBP knockdown in IHH cells (Fig. 1G) and Caspase 3 cleavage was not observed (data not shown). These results indicate that inhibition of IHH proliferation upon ChREBP knockdown occurred in parallel to cell cycle arrest while apoptosis remained unaffected.
Normalization of hepatic ChREBP expression in L-G6pc-/- mice induces p53
activation and increased proliferation marker expression
We next evaluated the effect of hepatic ChREBP normalization in vivo, using short-hairpin mediated RNA interference (shRNA) in a liver-specific model for GSD Ia. Groups of wildtype (L-G6pc+/+) and hepatocyte-specific G6pc knockout (L-G6pc-/-) mice received shChREBP or shSCR by adeno-associated viral delivery as described previously (Chapter 2). Hepatic ChREBP knockdown normalized hepatic Chrebpβ mRNA levels in L-G6pc-/- mice to values observed in shSCR treated L-G6pc+/+ mice, while the expression of the ChREBP target genes Pklr, Fasn, Elovl6 and Scd1 also tended to reduce towards values observed in shSCR-treated L-G6pc+/+ mice (Fig. 2A). Gene Set Enrichment Analysis (GSEA) of RNA sequencing data from the livers of these mice showed a significant enrichment of p53 pathway genes in both L-G6pc+/+ and L-G6pc-/- mice that received shChREBP as compared to shSCR treated controls (Table 1 and Table S3, NES=1.54 and 1.54, respectively). Moreover, we observed that hepatic mRNA levels of the p53 target gene p21 were significantly increased in shChREBP-treated L-G6pc-/- mice only (Fig. 2B) and that both G1/S and G2/M checkpoint gene sets were significantly enriched in these animals as compared to shSCR treated controls (Table 1, NES= 1.64 and 1.95, respectively). The p53 pathway induction was paralleled by an increase in the reduced glutathione (GSH) levels and higher reduced/oxidized glutathione (GSH/GSSG) ratios in shChREBP-treated
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L-G6pc+/+ and L-G6pc-/- mice (Fig. 2C), indicative of enhanced antioxidative capacity upon hepatic ChREBP knockdown. GSEA showed that the nuclear factor erythroid 2-related factor 2 (NRF2) gene set was significantly enriched in shSCR-treated L-G6pc-/- versus L-G6pc+/+ mice, and that hepatic ChREBP knockdown in L-G6pc-/- mice further enhanced this enrichment (Table 1 and Table S3, NES= 1.73 and 2.26 respectively). Moreover, the number of Ki67 positive cells tended to increase upon hepatic ChREBP knockdown in L-G6pc-/- mice (Fig. 2D and Fig. S2), while the incidence of pH3 positive cells was higher in shChREBP treated L-G6pc+/+ and L-G6pc-/- mice (Fig. 2E and Fig. S2), and shChREBP-treated G6pc-/- livers showed more mitotic hepatocytes (Fig. 2F). Combined, these changes indicate enhanced oxidative stress, p53 activation and increased hepatocyte proliferation in the livers of shChREBP-treated L-G6pc-/- mice.
Figure 2. Normalization of hepatic ChREBP expression in L-G6pc-/- mice induces p53 activation and increases hepatocyte proliferation. A, relative mRNA expression of hepatic ChREBP and its target genes in livers of L-G6pc+/+ and L-G6pc-/- mice treated with shChREBP or shSCR. n=7-9. B, relative mRNA expression of p21 (Cdkn1a) in livers of L-G6pc+/+ and L-G6pc-/- mice treated with shChREBP or shSCR. C, relative levels of reduced glutathione (GSH), oxidized glutathione (GSSG) and the ratio of reduced glutathione/oxidized glutathione in livers of L-G6pc+/+ and L-G6pc-/- mice treated with shChREBP or shSCR. D, relative presence 102
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Figure 1. ChREBP knockdown in immortalized human hepatocytes (IHHs) reduces cell proliferation while inducing p53 activity and cell cycle arrest. A, relative mRNA and protein levels of ChREBP. Proteins were extracted from cells transfected with ChREBPα or ChREBPβ plasmids or siRNA of ChREBP or control. The bands of ChREBPα and ChREBPβ are indicated in the solid and dashed rectangles, respectively. Protein expression levels were expressed relative to β-actin. mRNA levels were expressed relative to B2M. B, relative mRNA levels of ChREBP target genes expressed relative to B2M. C, IHH cell growth curve after cells treated with siControl or siChREBP. D, cell apoptosis assessment with flow cytometry after treated with control or ChREBP siRNA for 48 hours. The right down corner window represents early apoptosis, while the right up corner window represents late apoptosis or cell death. E, cell cycle measurement with flow cytometry after treatment with control or ChREBP siRNA for 48 hours. F, relative mRNA expression of Ki67 as well as p53 target genes expressed relative to B2M. G, western blot of p21, phosphorylated-p53 and total Caspase 3. Protein expression levels were expressed relative to β-actin. Data in A, B, F and G is presented as box and-whisker plots indicating the sample minimum, lower quartile, median, upper quartile, and sample maximum. Data in C is presented as mean and SEM. n=3-4, *p<0.05, **p<0.01, ***p<0.001.
(TIGAR), E3 ubiquitin-protein ligase (MDM2) and C/EBP homology protein (CHOP) were all significantly increased in siChREBP-treated compared to control cells (Fig. 1F) and were paralleled by higher P21 and phospho-P53 protein expression levels (Fig. 1G). In agreement with unaltered numbers of Annexin V positive cells (Fig. 1D), Caspase 3 protein expression remained unaffected by ChREBP knockdown in IHH cells (Fig. 1G) and Caspase 3 cleavage was not observed (data not shown). These results indicate that inhibition of IHH proliferation upon ChREBP knockdown occurred in parallel to cell cycle arrest while apoptosis remained unaffected.
Normalization of hepatic ChREBP expression in L-G6pc-/- mice induces p53 activation and increased proliferation marker expression
We next evaluated the effect of hepatic ChREBP normalization in vivo, using short-hairpin mediated RNA interference (shRNA) in a liver-specific model for GSD Ia. Groups of wildtype (L-G6pc+/+) and hepatocyte-specific G6pc knockout (L-G6pc-/-) mice received shChREBP or shSCR by adeno-associated viral delivery as described previously (Chapter 2). Hepatic ChREBP knockdown normalized hepatic Chrebpβ mRNA levels in L-G6pc-/- mice to values observed in shSCR treated L-G6pc+/+ mice, while the expression of the ChREBP target genes Pklr, Fasn, Elovl6 and Scd1 also tended to reduce towards values observed in shSCR-treated L-G6pc+/+ mice (Fig. 2A). Gene Set Enrichment Analysis (GSEA) of RNA sequencing data from the livers of these mice showed a significant enrichment of p53 pathway genes in both L-G6pc+/+ and L-G6pc-/- mice that received shChREBP as compared to shSCR treated controls (Table 1 and Table S3, NES=1.54 and 1.54, respectively). Moreover, we observed that hepatic mRNA levels of the p53 target gene p21 were significantly increased in shChREBP-treated L-G6pc-/- mice only (Fig. 2B) and that both G1/S and G2/M checkpoint gene sets were significantly enriched in these animals as compared to shSCR treated controls (Table 1, NES= 1.64 and 1.95, respectively). The p53 pathway induction was paralleled by an increase in the reduced glutathione (GSH) levels and higher reduced/oxidized glutathione (GSH/GSSG) ratios in shChREBP-treated
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L-G6pc+/+ and L-G6pc-/- mice (Fig. 2C), indicative of enhanced antioxidative capacity upon hepatic ChREBP knockdown. GSEA showed that the nuclear factor erythroid 2-related factor 2 (NRF2) gene set was significantly enriched in shSCR-treated L-G6pc-/- versus L-G6pc+/+ mice, and that hepatic ChREBP knockdown in L-G6pc-/- mice further enhanced this enrichment (Table 1 and Table S3, NES= 1.73 and 2.26 respectively). Moreover, the number of Ki67 positive cells tended to increase upon hepatic ChREBP knockdown in L-G6pc-/- mice (Fig. 2D and Fig. S2), while the incidence of pH3 positive cells was higher in shChREBP treated L-G6pc+/+ and L-G6pc-/- mice (Fig. 2E and Fig. S2), and shChREBP-treated G6pc-/- livers showed more mitotic hepatocytes (Fig. 2F). Combined, these changes indicate enhanced oxidative stress, p53 activation and increased hepatocyte proliferation in the livers of shChREBP-treated L-G6pc-/- mice.
Figure 2. Normalization of hepatic ChREBP expression in L-G6pc-/- mice induces p53
activation and increases hepatocyte proliferation. A, relative mRNA expression of hepatic
ChREBP and its target genes in livers of L-G6pc+/+ and L-G6pc-/- mice treated with shChREBP
or shSCR. n=7-9. B, relative mRNA expression of p21 (Cdkn1a) in livers of L-G6pc+/+ and
L-G6pc-/- mice treated with shChREBP or shSCR. C, relative levels of reduced glutathione
(GSH), oxidized glutathione (GSSG) and the ratio of reduced glutathione/oxidized glutathione
in livers of L-G6pc+/+ and L-G6pc-/- mice treated with shChREBP or shSCR. D, relative presence
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of Ki67 positive immunohistochemical staining in livers of L-G6pc+/+ and L-G6pc-/- mice
treated with shChREBP or shSCR. E, relative presence of pH3 positive immunohistochemical
staining in livers of L-G6pc+/+ and L-G6pc-/- mice treated with shChREBP or shSCR. F, relative
presence of mitotic figures in livers of L-G6pc+/+ and L-G6pc-/- mice treated with shChREBP or
shSCR. n=7-9. For D, E and F, scoring was performed in 5 microscopic fields at 200x or 400x
magnification, corrected for the average hepatocyte size of each individual mouse. Data in C,
D and E were normalized to the average values in the L-G6pc+/+/shSCR-treated group. Data is
presented as scatter dot plots with median values. $represents the comparison between
L-G6pc+/+ and L-G6pc-/- mice treated with shSCR. *represents the comparison between shSCR
and shChREBP. *p<0.05, **p<0.01, ***p<0.001, $p<0.05, $$p<0.01, $$$p<0.001.
Normalization of hepatic ChREBP expression in L-G6pc-/- mice induces hepatocyte death, DNA damage and chromosomal instability
The divergent effects of shChREBP treatment on p53 pathway activation and proliferation markers in mice prompted us to further investigate hepatocyte characteristics upon hepatic ChREBP knockdown in vivo. The livers of shChREBP treated L-G6pc-/- mice showed a significant increase in single cell death as compared to shSCR treated L-G6pc-/- and L-G6pc+/+ mice (Fig. 3A).
Figure 3. Normalization of hepatic ChREBP expression in L-G6pc-/- mice induces hepatocyte
death, DNA damage and chromosomal instability. A, relative presence of single cell death in
livers of L-G6pc+/+ and L-G6pc-/- mice treated with shChREBP or shSCR. B, relative presence of
positive cleaved Caspase 3 immunohistochemical staining in livers of L-G6pc+/+ and L-G6pc
-/-mice treated with shChREBP or shSCR. C, relative hepatic Caspase 3 activity in livers of
L-G6pc+/+ and L-G6pc-/- mice treated with shChREBP or shSCR. D, relative presence of
inflammatory foci in livers of L-G6pc+/+ and L-G6pc-/- mice treated with shChREBP or shSCR. E,
relative presence of positive γH2Ax immunohistochemical staining in livers of L-G6pc+/+ and
L-G6pc-/- mice treated with shChREBP or shSCR. n=7-9. For A, B, D and E scoring was
performed in 5 microscopic fields at 200x or 400x magnification. corrected for the average
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hepatocyte size of each individual mouse, and normalized to the average values in the
L-G6pc+/+/shSCR-treated group. Data is presented as scatter dot plots with median values.
$represents the comparison between L-G6pc+/+ and L-G6pc-/- mice treated with shSCR.
*represents the comparison between shSCR and shChREBP. *p<0.05, ***p<0.001, $p<0.05.
This was paralleled by a slight, non-significant increase in the number of cleaved Caspase 3 positive cells (Fig. 3B and Fig. S2) and a significant enrichment of genes associated with cellular apoptosis (Table 1, NES= 1.68) in shChREBP versus shSCR treated L-G6pc-/- mice (Fig. 3B), while total hepatic Caspase 3 activity remained unaltered in these animals (Fig. 3C). Hepatic ChREBP knockdown in L-G6pc-/- mice also led to an increase in the number of inflammatory foci (Fig. 3D) and resulted in an enrichment of inflammatory response genes (Table 1, NES=1.97). Finally, hepatic ChREBP knockdown increased the number of γH2AX positive cells in both L-G6pc+/+ and L-G6pc-/- livers (Fig. 3E and Fig. S2) and resulted in significant enrichments of the CIN29 gene set in these groups (Table 1, NES= 1.89 and 2.13, respectively).
Discussion
In the current study we investigated the potential oncogenic role of hepatic ChREBP. Knockdown of ChREBP expression induced p53-activation in vitro and in vivo. However, this was associated with cell cycle inhibition in cultured hepatocytes while it was paralleled by cell death, mitosis, DNA damage and chromosomal instability in livers of GSD Ia mice. These findings indicate that the oncogenic role of ChREBP in hepatocytes is highly context-dependent.
ChREBP knockdown differentially affected the expression of its target genes in immortalized human hepatocytes (IHHs) as compared to mouse liver in situ. While PKLR, ELOVL6 and SCD1 mRNA levels were reduced under both conditions, the expression of FASN and ACLY was only reduced upon ChREBP knockdown in vivo. We speculate that this discrepancy is partly related to the supraphysiological insulin concentration of the IHH culture medium used (20 mU/mL, corresponding to 16.5 ng/mL), which likely results in a strong activation of Sterol Regulatory Element Binding Protein 1c (SREBP-1c), a transcription factor that also controls FASN and ACLY transcription (38, 39). Moreover, as IHHs do not express glucokinase (GCK) (40), these cells likely exhibit reduced glucose sensitivity as compared to hepatocytes in vivo (10, 41), which may alter the kinetics and extent of glucose-mediated ChREBP activation as compared to L-G6pc-/- livers. We therefore propose that, as a result of combined insulin-mediated SREBP-1c activation and loss of glucose sensitivity, ChREBP knockdown in IHH cells only modestly affects the expression of genes that are regulated by both ChREBP and SREBP-1c.
Our results furthermore indicate that lowering ChREBP expression triggers p53 activation in both IHH cells and L-G6pc-/- hepatocytes, as evidenced by the induction of its target gene p21 under both conditions and the significant enrichment of the p53 gene set in the livers of shChREBP treated L-G6pc-/- mice. In IHH cells these changes were paralleled by G1 arrest, consistent with what was reported by Tong
