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Iron Overload in Hereditary Tyrosinemia Type 1 Induces Liver

Injury through the Sp1/Tfr2/Hepcidin Axis

Wen-Dai Bao, Yao Fan, Yue-Zhen Deng, Ling-Yun Long, Jing-jing Wang,

Dong-Xian Guan, Zhen-Yu Qian, Peng An, Yuan-Yuan Feng, Zhi-Ying He,

Xiao-Fan Wang, H. Phillip Koeffler, Ronggui Hu, Jianshe Wang, Xin Wang,

Fudi Wang, Jing-Jing Li, Dong Xie

Table of contents

Supplementary materials and methods…………………………………..2

Supplementary Fig. 1………………………………………………………...6

Supplementary Fig. 2………………………………………………………...7

Supplementary Fig. 3………………………………………………………...9

Supplementary Fig. 4………………………………………………………..10

Supplementary Fig. 5………………………………………………………..11

Supplementary Fig. 6………………………………………………………..12

Supplementary Table 1……………………………………………….……..14

Supplementary Table 2……………………………………………….……..15

Supplementary Table 3……………………………………………….……..16

References……………..……………………………………………….……..18

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Supplementary materials and methods

Hepatocyte isolation and treatment

Mouse primary hepatocytes were isolated by perusing livers using collagenase

type I (Worthington) in perfusion buffers. After digestion, the viable

hepatocytes were filtered through a cell strainer (BD Falcon) followed by two

centrifugations using cold DMEM medium supplemented with 10% FBS. The

purified hepatocytes were seeded in collagen-coated plates at 8×105 cells per

well in a 6-well plate.

For hepatocyte holo-transferrin (holo-Tf) treatment, the hepatocytes were

cultured in DMEM medium supplemented with 5% FBS and antibiotics for 3

hrs after isolation. Cells were treated with 0, 25 or 50µM holo-Tf

(Sigma-Aldrich) for 24 hrs in DMEM containing 2% FBS [4]. Hepatocytes were

washed with PBS, and harvested for RNA and protein extraction and analysis.

Reactive Oxygen Species (ROS) production of hepatocytes was detected

by ROS detection molecular probes CM-H2DCFDA according to the

manufacturer’s instruction (Invitrogen). Cells were treated with antioxidants

6hrs after isolation. After 24hrs treatment, hepatocytes were incubated with

these dyes at 37°C for 20 minutes before measurement of ROS production.

Cells were collected, and the staining signals were detected by a flow

cytometer using excitation sources and filters appropriate for fluorescein

(FITC).

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Western Blot

Equal amounts of total cellular protein (15–30 mg) were resolved on

SDS-PAGE gels and transferred to polyvinylidene difluoride membranes

(Millipore, Bedford, MA). The membranes were blocked with 3% BSA for 1

hour and incubated overnight with primary antibodies. After washing, the

membranes were further incubated for 1 hour at room temperature with

corresponding horseradish peroxidase–conjugated secondary antibody then

washed 3 times. The immuno-reactive protein bands were visualized using an

enhanced chemiluminescence kit (Pierce Rockford, IL).

..

Preparation of Adenovirus

Full-length mouse open reading frame was amplified from a mouse liver cDNA

library by polymerase chain reaction (PCR) and inserted into the pcmv-myc

vector to obtain a myc tag first. The AdEasy Adenoviral Vector System

(Stratagene,Agilent Technologies) was used for the preparation of adenovirus,

the procedure was according to the manufacturer’s protocol. Sequences of

primers were available in Supplemental Materials. For animal experiment, we

intravenously delivered adenoviruses expressing Tfr2 or control virus, into 8

~10 week old female fah-/- mice after withdrawal of NTBC for one week.

Before the injection, we test the virus and minimize the amount of virus to

effectively overexpress the target gene but reduce the inflammation reaction.

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For mouse primary hepatocytes transfection, the isolated cells were

transfected after a 6 h attachment. Adenoviruses were applied in 1ml fresh

medium at multiplicity of infection (M.O.I) of 1~4 for each cell in a 6-well dish

for 6hrs, then replaced with 2ml fresh medium. 48hrs after transfection，the

hepatocytes were harvested for RNA and protein extraction and analysis.

Luciferase reporter Assay

Cells were plated at a subconfluent density and co-transfected with 0.05µg of

the reporter plasmid, 0.5 µg of expression vectors, and 0.05 µg of Renilla

luciferase, pRL-TK as an internal control for transfection efficiency. Cell lysates

were prepared 48h after transfection, and the reporter activity was measured

using the Dual-luciferase reporter assay system (Promega). Transfections

were performed in triplicate and repeated three times to ensure reproducibility.

Chromatin Immunoprecipitation Assay

Sp1 binding sites were present in the Tfr2 promoter region (Figure 4D).The

sp1 antibody specific for ChIP (Santa Cruz Biotechnology) was used in this

assay. The detailed procedures were performed as previously described

[3].Sequences of primers are available in Supplemental Experimental

Procedures.

Small interference RNA for Sp1

Duplexes of the small interference RNA for Sp1 (RNAi-Sp1) were synthesized

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and annealed (Shanghai GenePharma Co.,Ltd). The sequences of RNAi-Sp1

are 5-uugagucacccaaugagaatt-3 (forward) and

5-ttaacucaguggguuacucuu-3(reverse) [5], respectively, targeting both mouse

and human SP1 coding region relative to the translation start site. Huh7 and

hepa1-6 cells were transfected with Sp1 siRNA with lipofectamin RNAi max

reagent (invitrogen) according to the manufacturer’s protocol.

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Supplementary Fig. 1. Iron accumulated in Fah-/- male mice after NTBC

withdrawal.

(A) Tissue iron concentrations of different organs (liver, heart, kidney intestine,

spleen) and (B) serum iron parameters were measured in male fah-/- mice

after withdrawal of NTBC for 4 weeks (n=6) (C) HE staining and Perls’

Prussian blue, Dab-enhanced perI’s iron staining of liver sections from the

male fah-/- murine liver withdrawal of NTBC for 4 weeks.

Data are reported as mean ± SEM. Student’s t test: *p < 0.05; **p < 0.01; ***p

< 0.001

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Supplementary Fig. 2. Anti-oxidants could not rescue the

downregulation of hepcidin in fah-/- hepatocytes after NTBC withdrawal.

(A)ROS production in hepatocytes was examined by ROS-

sensitive fluorescence dye staining and calculated by FACS sorting.

Hepatocytes from wild-type mice were collected as control, hepatocytes from

fah-/- mice were treated with 1.0 mM NAC, 100μM PDTC or left untreated for

24 hours before staining.

(B) Hepatocytes from fah-/- mice withdrawal NTBC for one week were treated

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with 1.0 mM NAC, 100μM PDTC for 0, 8, 16, 24 hours or left untreated

(control), all the cells were collected simultaneously after treatment. Hepcidin

mRNA level was determined by qRT-PCR and normalized to 18S expression.

Data are reported as mean ± SEM. Student’s t test: *p < 0.05; **p < 0.01; ***p

< 0.001

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Supplementary Fig. 3. Add-back of NTBC restored Tfr2 expression in

fah-/- murine liver

(A) Protein level of Tfr2 in fah-/- murine liver of three groups: the control mice

with NTBC, the mice with one week of NTBC withdrawal and the mice with one

week add-back of NTBC after withdrawal (n=5). (B) Relative mRNA expression

level of Tfr2 in these mice.

Data are reported as mean ± SEM. Student’s t test: *p < 0.05; **p < 0.01; ***p

< 0.001

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Supplementary Fig. 4. Tfr2 remained suppressed in fah-/- murine liver on

low iron diet following NTBC withdrawal

(A) Protein level of Tfr2 in fah-/- murine liver of three groups: the control mice

with NTBC, the mice with control food after four week of NTBC withdrawal and

the mice with low-iron food after four week of NTBC withdrawal (n=5). (B)

Relative mRNA expression level of Tfr2 in these mice.

Data are reported as mean ± SEM. Student’s t test: *p < 0.05; **p < 0.01; ***p

< 0.001

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Supplementary Fig. 5. Iron status in fah-/- mice could not affect kidney

function.

(A and B) Crea(CR) and blood urea nitrogen (BUN) were determined as the

serum markers of kidney function in (A) fah-/- mice either with low iron or

control food intake after withdrawal of NTBC for 4 weeks and (B) in hfe-/-

×fah-/- , hjv-/-×fah-/- or fah-/- mice after withdrawal of NTBC for 5 weeks.

Data are reported as mean ± SEM. Student’s t test: *p < 0.05; **p < 0.01; ***p

< 0.001

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Supplementary Fig. 6. Ros related genes alterations were restored by

low-iron diet, while the iron status in fah-/- mice could not affect tyrosine

catabolism genes expression.

(A~D) Relative mRNA expression level of (A) TAT, (B) HPD, (C) HGD, (D)

MaaI in fah-/- murine liver of three groups: the fah-/- mice with NTBC, the mice

with control food after four week of NTBC withdrawal and the mice with

low-iron food after four week of NTBC withdrawal (n=5). (E~H) Relative mRNA

expression level of (E) Nrf2, (F) Gsta1, (G) Gsta2, (H) Nqo1 in these mice. (I)

Protein levels of Nrf2 in these mice. (J) Total glutathione levels were measured

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in these mice.

Data are reported as mean ± SEM. Student’s t test: *p < 0.05; **p < 0.01; ***p

< 0.001

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Supplementary Table 1. Serum iron of the HT1 patient

Serum iron(μg/dl)

Normal children

1 54.01±5.28

2 49.31±9.39

3 42.25±4.69

HT1 child 1 72.77±3.13

Serum iron were measured in the serum of the HT1 child patient and three

healthy children collected at the same day.

All samples were measured in triplicate for three times. Data are reported as

mean ± SEM,

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Supplementary Table 2. List of Primers Used for Plasmids Construct

Plasmid Gene primers

Pcmv-myc Mouse Tfr2

CCGGAATTCggATGGAGCAACGTTGGGGTCTA

CGGGGTACCTCAAAAGTTATTGTCAATGTTC

pcDNA3.1 Mouse Sp1

CGGGGTACCATGAGCGACCAAGATCACTC

CCGCTCGAGGAAACCATTGCCACTGATATTA

pAd-track-CMV Mouse Tfr2

GGAAGATCTATGGCATCAATGCAGAAGCT

CCGCTCGAGTCAAAAGTTATTGTCAATGTTCC

Mouse Sp1

CGGGGTACCATGAGCGACCAAGATCACTC

CCCAAGCTTTCAATGGTGATGGTGATGATGA

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Supplementary Table 3 List of Primers Used for qRT-PCR and CHIP

Gene Forward primer Reverse primer

18s-mus GTAACCCGTTGAACCCCATT CCATCCAATCGGTAGTAGCG

Hepcidin-mus AGAGCTGCAGCCTTTGCAC GAGGTCAGGATGTGGCTCTA

Tfr2-mus CCGCTATGGAGACGTGGTT TGGCGACACATACTGGGGACAG

Tfr1-mus CATGAGGGAAATCAATGATCG TA GCCCCAGAAGATATGTCGGAA

Fpn-mus TTGCAGGAGTCATTGCTGCTA TGGAGTTCTGCACACCATTGAT

Hfe-mus AGATCCGTGCCAAACAGAAC GGCGAGTCACTTTCACCAA

Hjv-mus TCTGACCTGAGTGAGACTGC GATGATGAGCGCTCCTACCTA

L-ferritin mus CCATCTGACCAACCTCCGC CGCTCAAAGAGATACTCGCC

HPD mus TCGGTGACCTTCTGGGTTG CAAAGCCCATCTTGTTGCAGTA

TAT mus TGCTGGATGTTCGCGTCAATA CGGCTTCACCTTCATGTTGTC

HGD mus ATTTGGGAATGAGTGTGCTTCA CTCAGCGTAGAGGTTGTAGGG

MaaI mus CCGGGAAGCCTATCCTCTACT CCGGGAAGCCTATCCTCTACT

Nrf2 mus TCTTGGAGTAAGTCGAGAAGTGT GTTGAAACTGAGCGAAAAAGGC

Gsta1 mus GACTGTGAGCTGAGTGGAGAAGAA CCGGCCATTGCAGCAA

Gsta2 mus CTCTTCAAACTCCACCCCTG TGGAGAAGAAGCCAGGACTC

Nqo1 mus AATGGGCCAGTACAATCAGG CCAGCCCTAAGGATCTCTCC

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Sp1-mus GTGGTGATGGGATACATGATGAGA CAAGTGTGCTCGGAGATGTGA

Actin-hum GATCATTGCTCCTCCTGAGC ACTCCTGCTTGCTGATCCAC

Sp1-hum CCAGACCATTAACCTCAGTGCAT CTGGGCTGTTTTCTCCTTCCT

Tfr2-hum GGACGGTGGTGACTTTGGAA TGCTTCAGGACACTCTCAATGAG

tfr2-sp1-chip-1 GAAAAAAGAAGGGAAGAGGAAGG TCCAATCTCTTGTTCCCGACTC

tfr2-sp1-chip-2 GCTCAACCATCACACACAAACAC CTCGGCTTTCTACTTGCCTTCAT

tfr2-sp1-chip-3 CTTTTCTCAGGGCACATTCCAG GGTAAGAGAGAAGAGGGTAA

TGGGA

tfr2-sp1-chip-4 AATTTCTTAGGTTTTAGTGGGGTCA GGCCACACCCACTAGAGCAC

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