Relative transcriptional activities of SAA1 promoters polymorphic atposition 713(T/C): Potential association between increasedtranscription and amyloidosis

MASATO MORIGUCHI1,2, HIROTAKA KANEKO1, CHIHIRO TERAI1, YUMI KOSEKI1,

HIROSHI KAJIYAMA1, SHINICHI INADA3, YUTAKA KITAMURA1, &

NAOYUKI KAMATANI1

1Institute of Rheumatology, Tokyo Women’s Medical University, Tokyo, Japan, 2Omiya Medical Center, Jichi Medical

School, Saitama, Japan, and 3Tokyo Metropolitan Ohtsuka Hospital, Tokyo, Japan

(Received 2 March 2004; accepted 21 September 2004)

Keywords: Serum amyloid A, AA-amyloidosis, polymorphism, luciferase reporter gene assay

Abbreviations: RA, rheumatoid arthritis; FMF, familial Mediterranean fever; SAA, serum amyloid A; SNP, singlenucleotide polymorphism; A-SAA, acute phase serum amyloid A

AbstractThe risk associated with the serum amyloid A (SAA) 1 gene and developing AA-amyloidosis is still controversial. In familialMediterranean fever or Caucasoid rheumatoid arthritis (RA), the SAA1.1 allele is a risk factor for the development of AA-amyloidosis. However, individuals with the SAA1.3 allele are susceptible to AA-amyloidosis in the Japanese RA population,but those with the SAA1.1 are not. Previous reports have indicated that the7 13T/C single nucleotide polymorphism (SNP)at the 5’-flanking region of SAA1 appears to be a better marker of AA-amyloidosis than the exon-3 based haplotype, i.e.,SAA1.1 or SAA1.3, in both Japanese and American Caucasian populations. So far, it is unknown why the 7 13T SNPincreases the amyloidogenicity of the patients. In the present study, a luciferase reporter gene assay showed that thetranscriptional activity of the SAA1 having the 7 13T-containing promoter was significantly higher than activities of thosewith 7 13C-containing promoters (Fisher’s protected least significance difference test). We suggest that having the 7 13TSNP in the SAA1 promoter correlates with the amyloidogenicity in part as a result of this increased transcriptional activity.

Introduction

Reactive AA-amyloidosis is a serious complication of

both rheumatoid arthritis (RA) and familial Medi-

terranean fever (FMF). The precursor protein in

AA-amyloidosis is serum amyloid A (SAA), which is

an acute phase protein mainly synthesized in the liver

by stimulation with proinflammatory cytokines, such

as IL-1, TNF-a and IL-6 [1]. The SAA1 and SAA2

genes on the short arm of chromosome 11 code acute

phase SAA (A-SAA) proteins, SAA1 and SAA2,

respectively. While an MEFV missense mutation,

M694V, is associated with a severe course of FMF

and developing AA-amyloidosis [2–8], the risk

associated with the SAA1 gene and developing AA-

amyloidosis is still controversial. Although homo-

zygosity for SAA1.1 is reported to be a risk factor for

the development of AA-amyloidosis in patients with

FMF [9–11] and in Caucasians with RA [12,13], this

is not the case in the Japanese RA population. The

SAA1.3 allele and not the SAA1.1 allele was closely

associated with AA-amyloidosis in Japanese RA

patients [14–17], with the SAA1.1 allele frequency

being significantly less in Japanese AA-amyloidosis

patients than in controls. In order to resolve this

discrepancy, we searched for new single nucleotide

polymorphisms (SNPs) in the 5’-flanking region of

the SAA1 sequence. We found one SNP, 7 13T/C

SNP, which correlated with the presence of AA-

amyloidosis in Japanese RA patients more strongly

than the SAA1.3 allele [18]. In that study, we

suggested that the 7 13T/C SNP represented a

superior marker of AA-amyloidosis than the exon-3

based haplotype SAA1.1 or SAA1.3. Recently, two

Correspondence: Dr. Masato Moriguchi, Omiya Medical Center, Jichi Medical School, 1-847 Amanuma-cho, Omiya-ku, Saitama-shi, 330-8503, Japan.

Tel: 81-48-647-2111. Fax: 81-48-648-5188. E-mail: mmorigu@omiya.jichi.ac.jp

Amyloid, March 2005; 12(1): 26–32

ISSN 1350-6129 print/ISSN 1744-2818 online ª 2005 Taylor & Francis Group Ltd

DOI: 10.1080/13506120500032394

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different papers were published regarding the asso-

ciation between the 7 13T/C SNP and AA-

amyloidosis. Gershoni-Baruch et al. examined the

7 13T/C SNP in a population of FMF patients in

Israel [11] and found no association between the

7 13T/C SNP and the susceptibility to renal

amyloidosis. The other study, by Yamada et al.,

established a positive association between the 7 13T

SNP and AA-amyloidosis in both American Cauca-

sian and Japanese populations [13]. In that paper,

they found significantly higher allele frequencies of

both SAA1.1 and the 7 13T SNP in American

Caucasian patients with AA-amyloidosis compared

to controls. In addition, the relation between the

7 13T SNP and AA-amyloidosis was much closer

than that between the SAA1.1 allele and AA-

amyloidosis in the American Caucasian population

(w2 = 12.30, p5 0.001 versus w2 = 7.33, p5 0.01,

respectively).

We suspect that functional differences between the

7 13T-containing SAA1 promoter and the 7 13C-

containing promoter affect the susceptibility to AA-

amyloidosis in both the American Caucasian and

Japanese populations. To elucidate this issue, we

have compared the activity of the promoter between

the 7 13T-containing cis-regulating element and the

7 13C-containing one using a luciferase reporter

gene assay.

Materials and methods

Determination of the haplotypes of the SAA1 promoter

and SAA1 genotypes

Each SNP at 7 2A/G, 7 13T/C and 7 61C/G in the

SAA1 promoter region was determined in genomic

DNA samples from 157 Japanese individuals,

including 44 RA patients with AA-amyloidosis, 55

RA patients without AA-amyloidosis and 58 healthy

controls, according to methods previously described

[18]. The haplotype frequencies of SAA1 SNPs

containing 3 SNPs at the promoter region and 2

SNPs at exon-3 were determined by the LDSupport

program developed in our laboratory [19,20]. This

program was designed for haplotype typing using a

maximum likelihood estimation method based on

the expectation-maximization algorithm.

Cells and reagents

Human hepatoma cell line, HepG2, was purchased

from DAINIPPON PHARMACEUTICAL CO.,

LTD. (Osaka, Japan). The cells were maintained in

5% CO2 at 378C in Eagle’s minimum essential

medium (MEM) with 10% fetal bovine serum and

supplemented with 1 mM sodium pyruvate, 1%

MEM nonessential amino acids, 2% L-glutamine,

100 U/ml penicillin and 100 mg/ml streptomycin.

Human recombinant IL-1b and IL-6 were purchased

from R&D Systems (Minneapolis, MN).

SAA1 promoter-luciferase construct

Using primers 5’-GTG CAG TGG CGT GAT

TAT AG-3’ and 5’-GAA GAT CTT CGT GCT

GTA GCT GAG CTG CGG-3’, the PCR

products of the promoter region of SAA1 were

obtained from the patient’s genomic DNA. In-

formed consent was obtained from all DNA

donors. After digestion with Bgl II, the PCR

fragments (560 bp of the promoter region of

SAA1, 7 534 to + 26) were subcloned into the

Bgl II site of the pGL-3 basic vector, which has a

down stream luciferase (Promega Madison, WI).

The insertion direction was confirmed by DNA

sequencing. We prepared 4 types of SAA1 pro-

moter-luciferase constructs based on 3 SNPs, at

7 61, 7 13 and – 2, of the SAA1 promoter region,

and designated them the 7 13C1(C-C-G),

7 13C2(G-C-G), 7 13C3 (G-C-A) and 7 13T(C-

T-G) promoter constructs (Figure 1A).

Transient transfection and luciferase assay

HepG2 cells were transfected with 0.25 mg of the

SAA1 promoter-luciferase construct and 0.006 mgof the pRL-TK vector (Promega), using FuGENE6

(Roche, Indianapolis, IN) according to the manu-

facture’s instructions. At 12 h after transfection,

the cells were stimulated with IL-1b (10 ng/ml)

and IL-6 (10 ng/ml) and harvested in the provided

lysis buffer, 0, 4, 6, 8, 16 and 24 h later. After

centrifugation of the cell solutions, the super-

natants were assayed for Firefly and Renilla

luciferase activity, using a TD-20/20 Luminometer

(Promega). The Firefly luciferase activity was

normalized to Renilla luciferase activity.

The relative luciferase activity of the 7 13C2,

7 13C3 or 7 13T promoter was expressed as a ratio

to the luciferase activity of the 7 13C1 promoter.

Data were obtained in duplicate from each experi-

ment, which was repeated 3 times. Relative luciferase

activity data for cultures harvested at 6 h after

stimulation with cytokines were subjected to analysis

of variance (ANOVA), and the differences were

determined by the one-way ANOVA followed by

Fisher’s protected least significance difference

(PLSD) test and other methods of post-hoc compar-

isons (Dunnett’s test and Tukey’s tests). For the

non-parametric analyses of time course luciferase

activity data, we applied Friedman’s test and after-

wards ran the Nemenyi’s test for multiple

comparisons to check which of the 2 groups was

significant.

SNPs at SAA1 and transcriptional activities 27

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Figure 1. Comparison of transcriptional activity between the 713C-containing SAA1 promoter and the 713T-containing SAA1

promoter. As described in ‘‘Materials and methods,’’ HepG2 cells were cotransfected with the SAA1 promoter-luciferase construct and

pRL-TK. Twelve h after transfection, cells were stimulated with IL-1b (10 ng/ml) and IL-6 (10 ng/ml). Cells were harvested and tested for

Firefly and Renilla luciferase activity at 6 h (B) or the indicated times (C). The Firefly luciferase activity was normalized to the Renilla activity.

(A) Schematic of 4 SAA1 promoter-luciferase constructs generated from 4 haplotypes of SAA1 promoter based on 3 SNPs 761C/G,

713T/C and7 2 A/G. Each SAA1 promoter construct was inserted into the pGL3 basic vector. (B) Transcriptional function of each SAA1

promoter. Each relative luciferase activity of the7 13C2,713C3 or 713T promoter is expressed as a ratio to that of the 713C1 promoter.

Each bar represents the mean of triplicate relative luciferase activities calculated as above, whilst the error bar represents SD of 3

experiments. (C) Comparison of time course data for the transcriptional function of each promoter. X-axis indicates duration of stimulation

with IL-1b (10 ng/ml) and IL-6 (10 ng/ml). The relative luciferase activity at each time point was expressed as a ratio to that at the point 0-h

in each promoter construct. We used Friedman’s test to analyze the effect of allelic variances on the luciferase reporter gene activities. Each

bar represents the mean of triplicate experiments whilst the error bar represents SD.

28 M. Moriguchi et al.

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Results

Haplotype frequencies of SNPs in the SAA1 promoter

and exon-3

In the analysis of 157 Japanese, we found 4 major

haplotypes of the SAA1 promoter based on 3 SNPs

at 7 61, 7 13 and 7 2 of the SAA1 promoter

region, and designated them 7 13C1(C-C-G),

7 13C2(G-C-G), 7 13C3 (G-C-A) and 7 13T(C-

T-G). The SAA1 allelic variants were determined

using a combination of 2 SNPs (2995C/T and

3010C/T) from the exon-3 region of the SAA1 gene

as previously reported [14,16]. The combinations of

SNPs at 2995 and 3010 of SAA1.1, SAA1.3 and

SAA1.5 are T-C, C-C and C-T, respectively. The

7 13C1 and 7 13T promoters were found to be

mainly linked to SAA1.1 and SAA1.3, respectively,

and both the 7 13C2 and 7 13C3 promoters to

SAA1.5 (Table I). The 7 13T/SAA1.3 haplotype

was the most frequent of all haplotypes found in the

Japanese AA-amyloidosis population.

Luciferase reporter gene assay

To compare the transcriptional activities of the 4

haplotypes of the SAA1 promoter, i.e., the 7 13C1,

7 13C2, 7 13C3 and 7 13T promoters, we pre-

pared 4 luciferase reporter gene constructs, each

including one of the SAA1 promoters (Figure 1A).

A-SAA is mainly synthesized by the liver, so we

chose the hepatocellular carcinoma cell line HepG2

for transfection. These cells have been shown to

produce significant amounts of A-SAA after treat-

ment with IL-1 and IL-6, the principal cytokines

involved in the up-regulation of A-SAA production

[21].

The luciferase activity of each SAA1 promoter

reached a maximum 6 h after stimulation with IL-1band IL-6 in a preliminary experiment (data not

shown). As shown in Figure 1B, after 6-h stimulation

with cytokines the relative luciferase activities of the

7 13C1, 7 13C2, 7 13C3 and 7 13T constructs

were 1, 0.88+ 0.15, 0.61+ 0.13, and 1.31+ 0.25

(mean+SD), respectively. By one-way ANOVA, we

found significant differences in transcriptional activ-

ity among the 4 luciferase reporter gene constructs

(F=9.97, p5 0.01), and so we applied post-hoc

analyses to determine which of each two groups was

significant. The relative luciferase activity of the

7 13T haplotype was significantly higher than the

other 3 haplotypes by Fisher’s PLSD test (p-values

for 7 13T versus 7 13C1, 7 13C2, and 7 13C3

were 5 0.05, 5 0.02, and 5 0.001, respectively).

Additionally, the transcriptional activity of 7 13C1

was higher than that of 7 13C3 (p5 0.02). Other

post-hoc tests, Dunnett’s and Tukey’s tests, also

indicated significant differences (p5 0.05) between

7 13T versus 7 13C2 and 7 13T versus 7 13C3.

Figure 1C illustrates a time course of the relative

promoter activities of the 7 13C1, 7 13C2, 7 13C3

and the 7 13T haplotypes. In this experiment, we

stimulated each group of cells with IL-1 and IL-6 for

4, 8, 16, or 24 hours. For each promoter construct

the relative luciferase activity at each time point was

measured and expressed as a ratio to that at the 0-h

time point. We used Friedman’s test to analyze the

effect of allelic variances on the luciferase reporter

gene activities. The factor of allelic variances

influenced the luciferase activity with an overall

significance (Friedman’s w2 = 10.69 with 3 degrees of

freedom, p5 0.02), indicating that there were

significant differences in the transcriptional activities

among the different SNPs in the SAA1 promoter

region. Further analysis using Nemenyi’s test for

multiple comparisons determined that only the

difference between 7 13T and 7 13C3 was signifi-

cant (p5 0.05).

Discussion

It is clear that the genotypes at the SAA1 locus are

associated with a susceptibility to develop AA-

Table I. Haplotype frequencies of the SAA1 promoter linked to the exon-3 based SAA1 allele.

Promoter haplotype/SAA1 allele Rheumatoid Arthritis Control

Amyloid ( + ) Amyloid (7)

(Number of alleles) (88) (110) (116)

713C1/SAA1.1 6.1% 31.6% 29.7%

713C2/SAA1.5 20.2 26.6 20.3

713C3/SAA1.5 1.4 6.2 4.2

713T/SAA1.3 60.9 26.3 38.1

713T/SAA1.1 1.2 1.8 –

713T/SAA1.5 4.1 4.5 6.0

713C1/SAA1.3 1.2 0.9 –

713C2/SAA1.3 – – 0.9

Others 4.9 2.0 0.8

SNPs at SAA1 and transcriptional activities 29

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amyloidosis, although it appears that the susceptible

alleles differ amongst different ethnic populations

[9–17]. So far, there has been no reported study

indicating the mechanism whereby the allelic

variants at the SAA1 locus influence amyloidogeni-

city.

In cases of long-standing inflammatory diseases

like RA or FMF, amyloid A proteins derived from

the N-terminal of the A-SAA protein form amyloid

fibrils and deposit in the extra cellular region of

multiple organs. After a period of several years to

decades, the patient’s condition may be complicated

by multiple organ failure. Several processes, includ-

ing the production of the A-SAA protein, its

conversion to amyloid A protein, and the rate of

tissue deposition, are thought to correlate with the

susceptibility to AA-amyloidosis.

Our previous data suggested that an SNP in the 5’-

flanking region of SAA1, 7 13T/C, was more closely

associated with AA-amyloidosis than 2 SNPs in the

coding region (2995C/T and 3010C/T). Another

group also confirmed this association and its useful-

ness as a predictor of AA-amyloidosis in an

American Caucasian population [13]. We focused

on the transcription of SAA1 in the present study.In

luciferase reporter gene assays, we demonstrated that

the 7 13T promoter exhibited greater transcrip-

tional activity compared to the other cis-regulating

elements, 7 13C1, 7 13C2 and 7 13C3, after a 6-hr

stimulation with IL-1b and IL-6 (Figure 1B).

In the Japanese population, the promoter SNPs

7 13T and 7 13C1, in the 5’-flanking region of

SAA1, exhibit strong linkage disequilibrium to the

SAA1.3 and SAA1.1 alleles, respectively, and both

7 13C2 and 7 13C3 are linked to the SAA1.5 allele

as shown in Table I. Previous studies have demon-

strated that, in different ethnic populations, the

SAA1.1 or SAA1.3 allele exhibits stronger amyloi-

dogenicity than SAA1.5 [9–17]. This may result

from a greater transcriptional activity of the promo-

ters, SAA1.1 or SAA1.3 (i.e., 7 13C1 or 7 13T)

compared to that of SAA1.5 (i.e., 7 13C2 or

7 13C3).

According to Yamada et al., the serum A-SAA

protein concentration appeared to vary among the

3 SAA1 genotypes [16]. The serum concentration

ratios of A-SAA to C-reactive protein (CRP) were

significantly higher in the population carrying the

SAA1.5 genotype than in either the SAA1.1 or

SAA1.3 genotype population. The data from our

luciferase reporter gene assay seem inconsistent

with Yamada’s results. However, an increased

transcriptional activity does not necessarily result

in an elevated serum protein concentration as

many factors, such as mRNA stability, A-SAA

protein degradation and transfer to extra vascular

regions, may influence the serum concentration of

the A-SAA protein. The serum concentration of

the A-SAA protein may depend upon the rate of

protein production and metabolism. Therefore,

even if the SAA1.1 or SAA1.3 protein can be

produced more readily than the SAA1.5 protein, it

may be cleared more rapidly so that the serum

concentration might settle at lower levels, as were

found in Yamada’s report. In fact, Yamada’s latest

paper showed that recombinant human SAA1.5

disappeared from plasma more slowly than the

other isotypes, SAA1.1 and SAA1.3 [22]. If faster

clearance of SAA correlates with higher production

of amyloid A fibrils, individuals with the SAA1.5

allele should be less susceptible to AA-amyloidosis

than those with other alleles. Therefore, our results

from the luciferase reporter gene assays and

Yamada’s results would support a hypothesis that

greater production and faster clearance of SAA1

are associated with the susceptibility to AA-

amyloidosis.

We previously demonstrated a significant negative

correlation between the number of SAA1.3 (SAA1g)alleles and the duration of RA, prior to a diagnosis of

AA-amyloidosis. Conversely, a significant positive

correlation was found between the number of

SAA1.3 alleles and the mean CRP level before a

diagnosis of AA-amyloidosis [15]. In addition, we

found that the SAA1.3 allele was associated with the

clinical severity of AA-amyloidosis, including symp-

toms of hypoalbuminemia and massive proteinuria

compared to other SAA1 alleles [17]. These clinical

findings indicated that the SAA1.3 allele possibly

results in enhanced inflammatory responses in

patients with AA-amyloidosis. As most of the

SAA1.3 genotypes carry the 7 13T SNP at the

SAA1 promoter region (Table I), the 7 13T SNP

possibly accelerates response of many cytokines

through an increase in transcriptional activity and

production of A-SAA. In fact, previous studies have

provided data indicating that A-SAA has cytokine-

like abilities, such as induction of synthesis of matrix

metalloproteinases [23], IL-1b [24,25], IL-1 recep-

tor antagonist, soluble TNF-a type II receptor [24],

TNF-a [25] and IL-8 [25,26]. Recently, He et al.

showed that A-SAA stimulates NF-kB activation,

phosphorylation of ERK1/2 and p38, and transcrip-

tion of the IL-8 gene through a G protein-coupled

receptor, FPRL1/LXA4R [26]. It is possible that the

SAA1 promoter having the7 13T SNP increases the

production of A-SAA in an autocrine manner by

NF-kB activation, through the FPRL1/LXA4R

signaling pathway. Although the difference in the

transcriptional activity between the 7 13T and

7 13C promoters, especially 7 13T versus

7 13C1, seems small, an accumulative effect on the

long-standing inflammation of RA may result in a

large difference in amyloidogenicity.

30 M. Moriguchi et al.

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In conclusion, we suggest that the susceptibility to

AA-amyloidosis may be modulated by the 7 13T

SNP of the SAA1 promoter as a result of the

increased transcriptional activity of the SAA1 gene.

So far, we cannot deny the possibility that the SNPs

in the coding region of SAA1 may correlate with the

amyloidogenicity through a different property of the

SAA1 isotypes. Further studies are necessary to

determine which are the predominant SNPs, coding

or non-coding, associated with the susceptibility to

AA-amyloidosis.

Acknowledgments

This work was supported by grants from the Ministry

of Education, Science, and Culture of Japan

(No.13670479 and 15591066).

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