BIOCHEMICAL AND BIOPHYSICAL RESEARCH COMMUNICATIONS
236, 71–74 (1997)
ARTICLE NO.
RC976906
Increase in Ref-1 mRNA and Protein by Thyrotropin
in Rat Thyroid FRTL-5 Cells
Toshi Asai, Fukushi Kambe, Toyone Kikumori, and Hisao Seo
Department of Endocrinology and Metabolism, Division of Molecular and Cellular Adaptation, Research Institute
of Environmental Medicine, Nagoya University, Nagoya 464-01, Japan
Received May 29, 1997
tivity by reducing the conserved cysteine (10, 11). It
Thyrotropin (TSH) induces the expression of fos and
has been reported that Ref-1 is a major reductant for
jun family genes in thyroid cells. The DNA-binding ac-
AP-1 in HeLa cells (11), because AP-1 binding activity
tivity of these gene products (AP-1) has been shown to
was extremely decreased when Ref-1 protein in the cell
be enhanced by ubiquitous nuclear redox factor-1
extract was depleted by immunoprecipitation using
(Ref-1). We thus examined whether TSH regulates Ref-
specific antibody. Recently, Yao et al showed that Ref-1
1 gene expression in rat thyroid FRTL-5 cells. North-
and AP-1 were simultaneously increased in the human
ern blot analysis revealed that the abundance of Ref-
HT29 colon cancer cells when they were exposed to
1 mRNA significantly increased within 3 hours after
hypoxia (12). We thus examined whether Ref-1 gene
TSH followed by a sustained increase until 12 hours.
expression is regulated by TSH or not, using a differen-
The increase was also induced by treatment with for-
tiated rat thyroid cell line FRTL-5.
skolin but not in the presence of cycloheximide, indi-
cating that the TSH effect on Ref-1 mRNA is mediated
by intracellular cAMP and requires de novo protein
MATERIALS AND METHODS
synthesis. Consistent with the elevation of the mRNA
level, Western blot analysis showed an increase in Ref-
Cell culture. FRTL-5 cells (ATCC CRL8305) were cultured in
1 protein 3 hours after TSH. The level continued to
Coon’s modified Ham’s F-12 medium (GIBCO-BRL, Gaithersburg,
increase until 12 hours. These results suggested that
MD) supplemented with 5% heat-inactivated calf serum (GIBCO-
increased Ref-1 by TSH might regulate the binding ac-
BRL) and six hormonesincludinghydrocortisone(10nM),transferrin
tivity of AP-1 in thyroid cells. Considering that Ref-1
(5
m
g/ml), somatostatin (10 ng/ml), glycyl-L-histidyl-L-lysine acetate
(10 ng/ml), bovine TSH (1 mU/ml) and insulin (10
m
g/ml) (13). After
also has a DNA repair function, Ref-1 may play dual
the cells reached to near confluency, the medium was replaced with
roles in gene regulation and DNA repair processes in
that containing no TSH, no insulin and 0.5 % serum (basal medium).
thyroid cells.
᭧ 1997 Academic Press
The cells were cultured in the medium for 4 days. The cells were
then incubated with 1 mU/ml TSH or 10
m
M forskolin for various
length of time and harvested for Northern blot and Western blot
analyses. In an experiment using cycloheximide (CHX, Wako, Tokyo,
Japan), it was added to the media at a concentration of 10
m
g/ml 15
Thyrotropin (TSH) induces the expression of c-fos
min before TSH treatment.
and c-jun genes in thyroid follicular cells (1-4). Our
Northern blot analysis. Total RNA was prepared by the acid gua-
previous study has demonstrated that TSH increases
nidinium thiocyanate-phenol-chloroform-extraction method (14).
the expression of not only c-fos and c-jun genes but also
The detailed procedures for Northern blotting were described in our
other fos and jun family genes such as fra-1, fra-2,
previous report (15). After fractionation of 15
m
g of total RNA on 0.8
fosB and junB with a differential time course (5). The
% agarose gel, the gel was stained with ethidium bromide. The RNA
was then transferred onto GeneScreen Plus membrane (Du Pont-
activator protein-1 (AP-1) consists of homodimer or het-
New England Nuclear, Boston, MA). The amounts of 28S and 18S
erodimer among fos and jun family gene products (6).
ribosomal RNAs in each lane were estimated from the photograph
It binds to an AP-1 site in a regulatory region of many
of the membrane taken under ultraviolet light (wave length: 320
vertebrate genes and regulates their expression (7).
nm). The membrane was hybridized with a rat Ref-1 cDNA probe
The DNA-binding activity of AP-1 is regulated by a
labeled with [
a
-
32
P]dCTP (New England Nuclear) using random
primed labeling kit (Boehringer Mannheim, Germany). The probe
posttranslational mechanism involving redox (8). Re-
contains a full coding sequence of rat Ref-1 (APEX nuclease) cDNA
dox regulation occurs through a conserved cysteine res-
which was a gift from Dr. Seki, Okayama University Medical School,
idue located in the DNA-binding domain of AP-1 (8). A
Okayama, Japan. After hybridization, the membrane was washed
ubiquitous nuclear redox factor-1 (Ref-1), also known
and exposed to XAR-5 film (Eastman Kodak, Rochester, NY) at 080
C with an intensifying screen. Quantitative analysis of the density
as APEX nuclease (9), increases the DNA-binding ac-
0006-291X/97 $25.00
Copyright ᭧ 1997 by Academic Press
All rights of reproduction in any form reserved.
71
AID BBRC 6906 / 6931$$$281
06-24-97 11:30:41 bbrcg AP: BBRC