2D DIGE proteomic analysis highlights delayed postnatal repression of α-fetoprotein expression in homocystinuria model mice.

Cystathionine β-synthase-deficient (Cbs (-/-)) mice, an animal model for homocystinuria, exhibit hepatic steatosis and juvenile semilethality via as yet unknown mechanisms. The plasma protein profile of Cbs (-/-) mice was investigated by proteomic analysis using two-dimensional difference gel electrophoresis and matrix-assisted laser desorption/ionization-time of flight/mass spectrometry. We found hyperaccumulation of α-fetoprotein (AFP) and downregulation of most other plasma proteins. AFP was highly expressed in fetal liver, but its expression declined dramatically via transcriptional repression after birth in both wild-type and Cbs (-/-) mice. However, the repression was delayed in Cbs (-/-) mice, causing high postnatal AFP levels, which may relate to transcriptional repression of most plasma proteins originating from liver and the observed hepatic dysfunction.

Introduction

Elevated levels of plasma homocysteine are an independent risk factor for atherosclerotic cardiovascular diseases, stroke, peripheral arterial occlusive diseases, and venous thrombosis [1]. Hyperhomocysteinemia is caused by several genetic defects, but mainly by deficiency of cystathionine β-synthase (CBS; EC 4.2.1.22). CBS-deficient homocystinuria patients (MIM 236200) exhibit various severe clinical manifestations including thromboembolism, mental retardation, osteoporosis, and skeletal abnormalities. The molecular mechanisms by which accumulated homocysteine may promote such diseases have been the focus of numerous investigations. Endothelial dysfunction appears to play a key role in cardiovascular diseases [2], but the pathogenesis of hepatic steatosis, a sporadic feature in CBS-deficient patients [1,3], remains to be clarified. It is notable that plasma homocysteine levels are elevated in patients with non-alcoholic fatty liver disease (NAFLD) [4].

A genetic model with targeted deletion of the Cbs gene was generated in 1995 [5] and has subsequently been widely used in homocysteine-related research. Homozygous Cbs−/− mice develop fatty liver at a juvenile age (∼2 weeks old) [5,6] and display an abnormal lipoprotein profile [7], but a few escape and fortunately survive beyond this age [8]. This study examined the plasma protein profile of Cbs−/− mice using proteomic analysis with fluorescent two-dimensional difference gel electrophoresis (2D DIGE) to gain insight into the molecular background of hepatic steatosis. For comparison, we utilized mice lacking cystathionine γ-lyase (CTH, also known as CSE; EC 4.4.1.1), which also display homocysteinemia but are free of obvious abnormalities (such as fatty liver) [9]. Here, we found hyperaccumulation of α-fetoprotein (AFP) in the plasma and fatty liver of Cbs−/− mice but not of Cth−/− mice.

Materials and methods

Animals

Heterozygous Cbs+/− mice in a C57BL/6J background (B6.129P2-Cbs/J) were obtained from the Jackson Laboratory (Bar Harbor, ME, USA). They were further backcrossed for 12 generations (N12) with C57BL/6JJcl (Jcl: Japan Clea, Tokyo, Japan) [8]. Heterozygous Cth+/− mice were generated by our group [9] and backcrossed for 10 generations (N10) with C57BL/6JJcl [10]. N12 Cbs+/− or N10 Cth+/− mice were bred to produce Cbs−/− or Cth−/− mice, and their age-matched progenies were analyzed comparatively. Mice were housed in an air-conditioned room kept on a 12-h dark/light cycle and allowed to free access to a standard dry rodent diet and water. All animal protocols were approved by the Animal Care Committee of Keio University (No. 09187-(4)).

Polyacrylamide gel electrophoresis (PAGE) and western blotting

Male mice were anesthetized with diethyl ether, and blood samples were collected from beating hearts of laparotomized mice, and then EDTA plasma (or serum) was prepared. Livers were removed quickly and frozen in liquid nitrogen until homogenization in ice-cold phosphate buffer containing Complete Mini, EDTA-free protease inhibitor cocktail (Roche Applied Science) [11]. Serum or liver homogenates (2.5 μg; quantified using Pierce BCA Protein Assay Kit, Thermo Scientific) were separated on a 10% SDS–PAGE gel and transferred to a polyvinylidene fluoride membrane (Immobilon-P, 0.45 μm, Millipore). The membranes were subjected to Western blotting analysis using goat anti-AFP antibody (1:200 dilution; sc-8108; Santa Cruz), anti-glyceraldehyde-3-phosphate dehydrogenase (GAPDH) antibody (1:1,000 dilution; #2118; Cell Signaling), horseradish peroxidase-conjugated anti-goat IgG (H+L) antibody (1:2,000 dilution; PI-9500; Vector Laboratories), and an ECL Prime detection system (GE Healthcare).

Measurement of plasma albumin levels

Plasma levels of albumin were measured using a Dri-Chem 7000i biochemistry analyzer with ALB-P slides (Fujifilm, Tokyo, Japan).

2D DIGE

Plasma (10 μL) was mixed with 90 μL lysis buffer (7 M urea, 2 M thiourea, 4% CHAPS, 1 mM PMSF, 1 mM Na3VO4). After adjusting the pH to 8.5 by adding 10 mM Tris–HCl (pH 8.5), 0.8 μL samples were labeled with 200 pmol of CyDye DIGE fluor, minimal labeling dye (Cy2, Cy3 or Cy5 [GE Healthcare]) at 4 °C for 30 min in the dark. The reaction was stopped by adding 0.5 μL of 10 mM lysine. Labeled samples were mixed with dithiothreitol (DTT) and immobilized pH gradient (IPG) buffer (final 1% each) at 4 °C for 10 min in the dark. The samples were immediately subjected to isoelectric focusing (IEF) in an Immobiline DryStrip (13 cm, pH 4–7 [GE Healthcare]) that were rehydrated for 20 h in a rehydration buffer (7 M urea, 2 M thiourea, 2% Triton X-100, 13 mM DTT, 2.5 mM acetic acid, 1% IPG buffer, and a trace amount of bromophenol blue) at 20 °C. IEF was performed using a CoolPhoreStar IPG-IEF Type-PX system (Anatech, Tokyo, Japan) in the following conditions: 500 V for 1.5 h, linear gradient from 500 V to 3500 V for 4.5 h, and finally 3500 V for 8 h at 20 °C. Once IEF was completed, the strips were equilibrated for 30 min in reducing buffer (50 mM Tris–HCl [pH 6.8], 6 M urea, 2% SDS, 30% [v/v] glycerol, 65 mM DTT and a trace amount of bromophenol blue), followed by an alkylating buffer (reducing buffer with 4.5% iodoacetamide instead of DTT) for an additional 15 min. The strips were sealed on the top of 10% PAGE gels using 0.5% low-melting-point agarose in a Tris–glycine electrophoresis buffer. The second dimension of protein separation was performed at a constant 200 V using an ERICA-S high-speed electrophoresis system (DRC, Tokyo, Japan) [12]. Gels were scanned using a Typhoon Trio image scanner (GE Healthcare).

Matrix-assisted laser desorption/ionization-time of flight/mass spectrometry (MALDI-TOF/MS) analysis

For silver staining, plasma (10 μL) was subjected to IEF and then SDS–PAGE without CyDye labeling. The gel was stained using a Silver Stain MS kit (Wako, Tokyo, Japan) in accordance with the manufacturer’s instructions. The gel pieces were excised, destained, washed twice with deionized water and four times with 50 mM ammonium bicarbonate: acetonitrile (1:1), and dehydrated once with acetonitrile. Then, the gel pieces were twice alternately rehydrated with 100 mM ammonium bicarbonate and dehydrated with acetonitrile, and dried by vacuum centrifugation. Protein samples were digested at 37 °C for 12 h with 5 μL of 0.02 μg/μL Sequencing Grade Modified Trypsin (Promega) dissolved in 25 mM ammonium bicarbonate. Peptides were extracted from the gels in 40 μL of 1% trifluoroacetic acid/50% acetonitrile solution by sonication. Samples were spotted onto a μFocus MALDI plate (900 μm, 384 circles, Hudson Surface Technology; Old Tappan, NJ, USA) with an equal volume of matrix solution, containing 10 mM α-cyano-4-hydroxycinnamic acid in 1% trifluoroacetic acid/50% acetonitrile. Positive ion mass spectra were obtained using an AXIMA-CFR Plus (Shimadzu, Kyoto, Japan) in a reflectron mode. MS spectra were acquired over a mass range of 700–4000 m/z and calibrated using peptide calibration standards (∼1,000–3,200 Da, Bruker Daltonics, Yokohama, Japan) [12].

Protein database search

Proteins were identified by matching the peptide mass fingerprint with the Swiss-Prot protein database using the MASCOT Search engine (Matrix Science, http://www.matrixscience.com). Database searches were carried out using the following parameters: taxonomy, Mus musculus; enzyme, trypsin; and allowing one missed cleavage. Carbamidomethylation was selected as a fixed modification, and the oxidation of methionine was allowed as a variable. The peptide mass tolerance was set at 0.5 Da and the significance threshold was set at P < 0.05 probability based values on Mowse scores (⩾55).

Quantitative polymerase chain reaction (qPCR)

Total RNA was isolated from the liver using TRI Reagent (Molecular Research Center, Cincinnati, Ohio, USA). Total RNA (1 μg) was used to produce first-strand cDNA with a ReverTra Ace qPCR RT Kit (Toyobo, Tokyo, Japan). A total of 10 ng of cDNA from each sample was amplified via qPCR using the SYBR Green Realtime PCR Master Mix (Toyobo), 18 primer sets (Table 1), and a CFX96 Touch Real-Time PCR Detection System (Bio-Rad) [11]. Each mRNA level was quantified using the comparative CT method with housekeeping gene hypoxanthine guanine phosphoribosyl transferase (Hprt) levels used for normalization, and the relative expression in wild-type mice was set at 1.

Table 1

Primer sets for qPCR.

Gene	Primer sequence	Size
Afp	5′-CAGGCACTGTCCAAGCAAAG-3′ (Forward)	218 bp
	5′-ATGAAAATGTCGGCCATTCC-3′ (Reverse)
Apoe	5′-TGCTGTTGGTCACATTGCTG-3′ (Forward)	145 bp
	5′-CTGCAGCTCTTCCTGGACCT-3′ (Reverse)
Apoa4	5′-AACAATGCCAAGGAGGCTGT-3′ (Forward)	132 bp
	5′-CTGCAGCTCTTCCTGGACCT-3′ (Reverse)
A2m	5′-CAGCAGCAGAAGGACAATGG-3′ (Forward)	171 bp
	5′-CAGGCAAAAGAGGGCATTTC-3′ (Reverse)
Cp	5′-AGCATTCAGCCAATGGGAGT-3′ (Forward)	148 bp
	5′-GTCCCATTTCTTTGGGGACA-3′ (Reverse)
Itih4	5′-CCCGATTTGCCCATACTGTT-3′ (Forward)	162 bp
	5′-CGGCCTTCTCTTTGACAACC-3′ (Reverse)
Hpx	5′-ATAGCTGGCCCATTGCTCAT-3′ (Forward)	161 bp
	5′-CTCCAGCCGCTTTGGATAAC-3′ (Reverse)
Kng1	5′-CACAGCAACTGTGGGGAAAA-3′ (Forward)	124 bp
	5′-TATGGCATGCACACAACCAA-3′ (Reverse)
Serpinc1	5′-AACCGCCTTTTTGGAGACAA-3′ (Forward)	120 bp
	5′-TCTGGATTGCTCCGGATTCT-3′ (Reverse)
Serpina1c	5′-TCTTCCTTCTGCCCGATGAT-3′ (Forward)	120 bp
	5′-GTCTGGGGAAGTGGATCTGG-3′ (Reverse)
Ahsg	5′-AGATTTCCCGGGCTCAAAAT-3′ (Forward)	156 bp
	5′-GCATGAGATTTGCCTTGCAG-3′ (Reverse)
Gc	5′-AGCACTCAGAGTCCCCTGCT-3′ (Forward)	127 bp
	5′-TCCTTAGCCGTTCTGCCAAT-3′ (Reverse)
Fgb	5′-CACCTCCATCAAGCCGTACA-3′ (Forward)	111 bp
	5′-TCCCATTTCCTGCCAAAGTC-3′ (Reverse)
Fgg	5′-CCTTCTTTGCTGCCTGCTTT-3′ (Forward)	172 bp
	5′-CGTTCGGAGATCATTGTCCA-3′ (Reverse)
Apoa1	5′-TGTGTATGTGGATGCGGTCA-3′ (Forward)	172 bp
	5′-ATCCCAGAAGTCCCGAGTCA-3′ (Reverse)
Alb	5′-AAAGACGTGTGTTGCCGATG-3′ (Forward)	122 bp
	5′-AGCAGTCAGCCAGTTCACCA-3′ (Reverse)
Zbtb20	5′-CCTTCCCTGCCTGAACTTTG-3′ (Forward)	176 bp
	5′-GCACGGAATTGCTGAAGTTG-3′ (Reverse)
Hprt	5′-GACTGATTATGGACAGGACTG-3′ (Forward)	211 bp
	5′-GACTGATCATTACAGTAGCTC-3′ (Reverse)

Statistical analysis

Data are expressed as means ± SD of independent samples (n as indicated). Statistical analyses were performed using unpaired two-tailed Student’s t-test, where P values ⩽0.05 were considered significant.

Results

Plasma protein profiling by 2D DIGE

Cbs−/− mice suffer from hepatic dysfunction/steatosis [6,7] and start to die from 2 weeks of age, and the majority die by 4 weeks [8], and thus we analyzed plasma protein profiles comparatively in 1-, 2-, and 4-week-old Cbs−/− and wild-type mice. Plasma protein concentrations were significantly lower in Cbs−/− mice (24.2 ± 4.5 μg/μL [n = 10] versus 37.2 ± 3.3 μg/μL [n = 8] in wild-type mice at 2 weeks of age; P < 0.001), and equal volumes of plasma samples were labeled fluorescently and separated by 2D DIGE (n = 3 for each genotype/age). The obtained fluorescent images indicated that most plasma protein spots were downregulated, whereas only few spots were upregulated in 2-week-old Cbs−/− mice (Fig. 1A), and such changes were not apparent in 1-week-old mice (Fig. 1B) but were generally maintained in 4-week-old mice (Fig. 1C).

Fig. 1

Altered plasma protein profiles in juvenile Cbs−/− mice revealed by 2D DIGE proteomic analysis. Plasma samples from wild-type (WT) and Cbs−/− male mice (n = 3 each) at 2 (A), 1 (B), and 4 (C) weeks of age were analyzed comparatively. Representative fluorescent image in which plasma samples from WT and Cbs−/− mouse plasma was pseudocolored in green and red, respectively, are presented with approximate isoelectric points (pI) and molecular weights (kDa).

MALDI-TOF/MS and Mascot search analyses identified a total of 48 spots for 16 protein species in the plasma samples (Table 2). AFP variants (spots 1a–d) and apolipoprotein E (spot 2) were highly expressed in 2-week-old Cbs−/− mouse plasma, compared with wild-type mouse plasma (Fig. 1A). In contrast, the expression levels of other major plasma proteins, α-2-macroglobulin (spots 4a–f), ceruloplasmin (spots 5a–e), inter α-trypsin inhibitor, heavy chain 4 (spots 6a–d), hemopexin (spots 7a–c), kininogen-1 (spots 8a–d), antithrombin-III (spot 9), α-1-antitrypsin 1-3 (spots 10a–d), α-2-HS-glycoprotein (spots 11a–c), vitamin D-binding protein (spot 12), fibrinogen β chain (spot 13), fibrinogen γ chain (spots 14a–d), and apolipoprotein A-I (spots 15b/c) were significantly downregulated in 2-week-old Cbs−/− mouse plasma (Fig. 1A). Most plasma proteins had several variant spots that dispersed with equal horizontal intervals in IEF; the spots had higher molecular weights as they shifted to lower (acidic) conditions, suggesting that they are redundantly sialylated and thus acidified. Expression of apolipoprotein A-IV (spot 3) and A-I variant (spot 15a) was similar between 2-week-old Cbs−/− and wild-type mouse samples (Fig. 1A), although the expression of both spots increased in 4-week-old Cbs−/− mouse samples (Fig. 1C). The amount of albumin, the most abundant protein in plasma, did not differ significantly (51.1–57.0% [n = 4–6]) among 1-, 2-, and 4-week-old wild-type and Cbs−/− mice (data not shown). Taken together, AFP was enriched whereas most other plasma proteins (except albumin) had decreased levels just before most Cbs−/− mice started to die.

Table 2

Differentially expressed plasma proteins between 2-week-old wild-type and Cbs−/− mice.

Spot ID	Uniprot ID	Unigene	Protein (up or down regulated in Cbs−/−)	Mascot score	Sequence coverage (%)	Peptide matches	MWcalc	pIcalc
1 a–d	P02772	Afp	α-fetoprotein (up)	218⁎	64⁎	36/104⁎	69,118	5.65
2	P08226	Apoe	Apolipoprotein E (up)	172	46	20/36	35,901	5.56
3	P06728	Apoa4	Apolipoprotein A-IV (up at 4 weeks)	145	61	23/58	45,001	5.34
4 a–f	Q61838	A2m	α-2-macroglobulin (down)	73⁎	16⁎	14/39⁎	167,116	6.24
5 a–e	Q61147	Cp	Ceruloplasmin (down)	150⁎	31⁎	26/47⁎	121,872	5.53
6 a–d	A6X935	Itih4	Inter α-trypsin inhibitor, heavy chain 4 (down)	100⁎	30⁎	25/79⁎	104,765	5.99
7 a–c	Q91X72	Hpx	Hemopexin (down)	115⁎	42⁎	18/84⁎	52,026	7.92
8 a–d	O08677	Kng1	Kininogen-1 (down)	112⁎	26⁎	18/59⁎	74,140	6.05
9	P32261	Serpinc1	Antithrombin-III (down)	92	35	16/49	52,484	6.10
10 a–d	Q00896	Serpina1c	α-1-antitrypsin 1–3 (down)	97⁎	37⁎	11/41⁎	45,966	5.25
11 a–c	P29699	Ahsg	α-2-HS-glycoprotein (down)	72⁎	33⁎	9/34⁎	38,100	6.04
12	P21614	Gc	Vitamin D-binding protein (down)	121	47	16/61	55,162	5.39
13	Q8K0E8	Fgb	Fibrinogen β chain (down)	174	60	35/120	55,402	6.68
14 a–d	Q8VCM7	Fgg	Fibrinogen γ chain (down)	212⁎	71⁎	24/45⁎	50,044	5.54
15 a–c	Q00623	Apoa1	Apolipoprotein A-I (down)	195⁎	50⁎	20/51⁎	30,597	5.51
16 a–c	P07724	Alb	Serum albumin (no change)	263⁎	60⁎	31/72⁎	70,700	5.75

A total 16 proteins identified from MALDI-TOF/MS analysis and Mascot searches are listed with their spot ID (in Fig. 1A), Uniprot ID, Unigene/protein names, Mascot score, sequence coverage, peptide matches, MW (molecular weight calculated from identified protein sequence), and pI (isoelectric point calculated from identified protein sequence).

Protein has some variant spots and the representative data from spots with the highest Mascot score are shown.

AFP expression in plasma (serum) and liver

Western blotting analysis using 2D PAGE identified nine AFP-positive spots around 80–90 kDa and pI 5.3–5.8 ranges, and all AFP spots were upregulated in 2-week-old Cbs−/− mouse plasma (Fig. 2A). We next examined AFP expression levels in the serum/liver of 2-week-old wild-type, Cbs heterozygous (Cbs−), Cbs−/−, Cth−, and Cth−/− mice. Serum and hepatic AFP levels were 3.5- and 7.3-fold, respectively, higher in Cbs−/− mice than in wild-type mice (Fig. 2B). AFP expression in Cth−/− mouse serum appeared slightly higher than in wild-type samples, but the difference was not significant (Fig. 2B). We further investigated AFP expression in serum and liver during postnatal development. AFP was highly expressed in embryonic liver and serum, but its expression was repressed postnatally (Fig. 2C). Compared with 2-week-old (P14.5: postnatal day 14.5) wild-type and Cbs+/− mice, in which their hepatic AFP expression was completely repressed and serum AFP expression was mostly repressed, substantial levels of AFP expression were observed in both the liver and serum of 2-week-old Cbs−/− mice (Fig. 2C).

Fig. 2

Increased α-fetoprotein (AFP) expression in 2-week-old Cbs−/− mouse plasma/serum/liver. (A) Two-dimensional PAGE/western blotting analysis of AFP variants in 2-week-old wild-type (WT) and Cbs−/− male mouse plasma (10 μL). (B) PAGE/western blotting analysis of AFP proteins in 2-week-old WT, (heterozygous) Cbs+/−, Cbs−/−, Cth+/−, and Cth−/− mouse serum and liver (2.5 μg protein per lane). As for liver samples, GAPDH expression was examined as a loading control. Band intensities of ∼79 kDa AFP proteins were densitometrically scanned and the relative values against the average AFP expression level (for serum) or the AFP/GAPDH ratio (for liver) in WT mouse samples were calculated. The representative band images are presented. Bar data show means ± SD (n = 3 each) and differences versus WT are significant at *P < 0.05 and **P < 0.01 by Student’s t-test. (C) PAGE/western blotting analysis of AFP proteins in embryonic day 15.5 (E15.5), postnatal day 0.5 (P0.5), P7.5, and P14.5 WT, Cbs+/−, and Cbs−/− mouse serum and liver (2.5 μg protein per lane). Postnatal repression of AFP expression was delayed in Cbs−/− mouse liver, and thus in Cbs−/− mouse serum.

Transcriptional regulation of plasma proteins

Hepatic mRNA expression of all identified genes was examined by qPCR (Fig. 3). Afp expression levels were similar between wild-type and Cbs−/− mice at P0.5. Afp expression in wild-type mice decreased dramatically at P14.5 (27.0-fold decrease from P0.5) and P28.5 (1,260-fold decrease), whereas that in Cbs−/− mice increased at P7.5 (2.33-fold increase) and then decreased at P14.5 (2.23-fold decrease) and P28.5 (15.6-fold decrease). As a result, Afp expression levels in Cbs−/− mice were 2.16-, 10.2-, and 68.3-fold higher than that in wild-type mice at P7.5, P14.5, and P28.5, respectively. Protein expression of apolipoprotein E was increased (Fig. 1A), but its mRNA expression level was decreased at P14.5 in Cbs−/− mice (Fig. 3). In addition, protein expression of apolipoprotein A-IV was higher in Cbs−/− mice only at P28.5 (Fig. 1C), but its mRNA expression levels were always higher that wild-type mice (Fig. 3). The expression levels of all other genes whose protein expression was significantly lower in Cbs−/− mouse plasma were all decreased in Cbs−/− mouse liver at P14.5 as well as at P28.5, indicating that decreased expression of most plasma proteins is attributable to their reduced mRNA expression in the liver. Xie et al. previously reported that postnatal Afp expression in mouse liver is repressed by the zinc finger protein ZBTB20 [13]. Hepatic Zbtb20 expression was lower in Cbs−/− mice, and therefore, ZBTB20 may be involved in delayed Afp repression (Fig. 3).

Fig. 3

Gene expression analysis of differentially expressed proteins and ZBTB20 in 2-week-old Cbs−/− mouse plasma by qPCR. Hepatic mRNA levels of α-fetoprotein (Afp), apolipoprotein E (Apoe), apolipoprotein A-IV (Apoa4), α-2-macroglobulin (A2m), ceruloplasmin (Cp), inter α-trypsin inhibitor, heavy chain 4 (itih4), hemopexin (Hpx), kininogen-1 (Kng1), antithrombin-III (Serpinc1), α-1-antitrypsin 1-3 (Serpina1c), α-2-HS-glycoprotein (Ahsg), vitamin D-binding protein (Gc), fibrinogen β chain (Fgb), fibrinogen γ chain (Fgg), apolipoprotein A-I (Apoa1), albumin (Alb), and Zbtb20 were analyzed by qPCR and normalized by hypoxanthine guanine phosphoribosyl transferase (Hprt) expression. The expression levels in wild-type mice (Wt) at postnatal day 0.5 (P0.5) (for Afp and Zbtb20) or P7.5 (for others) were set at 1. Data are shown as means ± SD (n = 4–6), and differences are significant in *P < 0.05, **P < 0.01 and ***P < 0.001 versus P0.5 (for Afp and Zbtb20) or P7.5 (for others); and #P < 0.05, ##P < 0.01 and ###P < 0.001 versus respective Wt samples by t-test.

Discussion

AFP is the major serum glycoprotein in the developing mammalian fetus that is produced by endodermal cells of the visceral yolk sac and the liver. The Afp gene is highly activated in the fetal liver but dramatically (∼10,000-fold) repressed soon after birth, and only trace amounts are detectable in adults [13]. Serum AFP elevation in adults is a well-known marker for hepatocellular carcinoma (HCC), yolk sac tumors, and acute/chronic hepatitis [14,15]. The characteristic machinery of Afp repression has attracted considerable attention from researchers interested in transcriptional regulation [13,16,17]; however, despite over 50 years of research since its first discovery in liver cancer, the physiological functions of AFP still remain obscure [14,18]. The main function of AFP is considered to be the extracellular transport of small molecules including estrogens, fatty acids, and bilirubin [19,20], but AFP-deficient mice develop normally and thus AFP is dispensable for embryonic development [21]. Meanwhile AFP is required for fertility in female mice [21] (i.e. protection of the developing female brain from masculinization/defeminization by estrogens [22]) and may play important immune regulatory roles [19,20]. This is the first demonstration of AFP accumulation during juvenile development in CBS-deficient mice, a homocystinuria model that is widely utilized for homocysteine-related research [5-8,12,23]. Our results may be clinically relevant because markedly lower Cbs expression in 120 HCC specimens compared with surrounding non-cancer liver cells was found to be associated with high tumor stage and serum AFP level [24], and serum AFP elevation was found in NAFLD patients [25].

Previous studies demonstrated that human AFP contains a single glycosylation site but its structure varies with developmental stage and disease state, probably by alternating glycosylation status [26,27]. Lens culinaris agglutinin (LCA)-reactive fraction of AFP (AFP-L3) has been considered as a more specific HCC marker [27]. In this study, we detected hyperaccumulation of all nine AFP variants in 2-week-old Cbs−/− mouse plasma compared with respective wild-type samples (Fig. 2A), which was attributable to delayed repression of Afp expression in the liver (Fig. 2C). In contrast, expression of most other major plasma proteins was suppressed (Table 2 and Fig. 1A), at least partly, via transcriptional repression (Fig. 3). One plausible explanation is that increased AFP may bind and hold multiple endogenous ligands required for transcriptional activation of such liver proteins. This is because AFP belongs to a three-domain albuminoid gene family that currently consists of four members (AFP, albumin, vitamin D-binding protein, and α-albumin) and binds steroids, fatty acids, bilirubin, retinoids, and heavy metals [19,20]. Indeed, vitamin D-binding protein, which binds/transports vitamin D and plays important roles in bone/calcium homeostasis [28], was downregulated in 2-week-old Cbs−/− mouse plasma (Table 2 and Fig. 1A). This downregulation could be related to osteoporosis and skeletal abnormalities found in Cbs−/− mice [29], although transgenic mice overexpressing human AFP were found to be generally normal [30], and hereditary persistence of AFP in two unrelated Japanese families exhibited no apparent phenotypes [31]. We reported previously about abnormal and decreased high-density lipoprotein contents in 2-week-old Cbs−/− mouse serum [7], which may be associated with decreased apolipoprotein A-I levels (Table 2 and Fig. 1A).

In conclusion, we found transcriptionally regulated hyperaccumulation of AFP in fatty liver and plasma of juvenile Cbs−/− mice. Mice lacking methionine adenosyltransferase 1A also displayed both fatty liver and AFP accumulation [32], but our Cth−− mice did not [9]; therefore, the methionine cycle/transsulfuration pathway may play important roles in epigenetic regulation of Afp.

Conflict of interest

The authors declare no competing financial interests.