BIOLOGY OF REPRODUCTION 57, 1056-1059 (1997)
Thioredoxin Messenger Ribonucleic Acid Is Regulated by Estradiol in the Rat Uterus' Lena Sahlin,2, 3 Arne Holmgren, 4 and Hakan Eriksson 3 Division for Reproductive Endocrinology, Department of Woman and Child Health, and Medical Nobel Institute for Biochemistry,4 Department of Biochemistry and Biophysics, Karolinska Institute, Stockholm, Sweden ABSTRACT Thioredoxin is a major cellular dithiol reductant with a large number of functions in electron transport and thiol redox control of enzymes and transcription factors. To investigate the expression and regulation of thioredoxin in the uterus, 35 rats were ovariectomized (OVX) and treated 14 days after surgery with either growth hormone (GH), dexamethasone (DEX), or estradiol (E, 2), or combinations of these, for 24 h. Thioredoxin mRNA levels were determined by solution hybridization. The animals receiving E, or a combination of E2 and DEX showed significantly increased thioredoxin mRNA levels, by 4-fold and 5-fold, respectively, as compared to the OVX control group. The GH or GH+DEX-treated groups did not display any difference in thioredoxin mRNA levels. Thioredoxin mRNA was also measured at different time points in uteri from OVX rats treated with daily E2 injections and was found to be transiently increased, with a maximum 48 h after the initiation of the E2 treatment. In contrast, the thioredoxin mRNA level in the liver of OVX rats was about 10-fold higher than in the uterus but remained unaffected by the different hormone treatments. We conclude that thioredoxin mRNA is expressed in the rat uterus, and up-regulated by E2 in a tissue-specific manner, with a maximum at 48 h after the initiation of hormone treatment. INTRODUCTION Thioredoxin (12 kDa), which contains two redox-active cysteine residues, has numerous functions such as electron transport to ribonucleotide reductase, regulation of protein activity by dithiol redox control, or defense against oxidative stress [1-3]. Together with thioredoxin reductase and NADPH, thioredoxin acts as a general protein disulfide reductase. Thus, thioredoxin and other members of the thioredoxin superfamily of proteins are of broad interest in cellular physiology and pathological conditions [4]. Thioredoxin is secreted from activated lymphocytes as a growth factor [5, 6] and operates as a co-stimulus of cytokine action [7]. The presence of thioredoxin in the human decidua and trophoblast has recently been demonstrated. In these tissues it was suggested to protect the fertilized egg and placental trophoblasts from the cytotoxic effects of oxygen radicals [8, 9]. Previously the expression of human thioredoxin (isolated as adult T-cell leukemia derived factor [ADF]) has been found to be very closely associated with the activity of steroid-producing cells in the human [10] and rat [11] ovary. In the human fetus, steroid-producing cells in the adrenal cortex, the ovary, and the testis have been shown to present strong immunohistochemical reacAccepted June 18, 1997. Received April 21, 1997. 'This work was supported by grants from Swedish Medical Research Council grants 03X-3972 (H.E.) and 13X-3529 (A.H.), Novo Nordisk Foundation (L.S.), and Karolinska Institutets Fonder. 2Correspondence: Lena Sahlin, Division for Reproductive Endocrinology, Karolinska Hospital, L5:01, S-171 76 Stockholm, Sweden. FAX: 468-517 734 85; e-mail:
[email protected]
tivity for thioredoxin [12]. In the present study, the regulation of thioredoxin expression in the rat uterus by E2 was examined, and its possible role in the estrogen mediated uterine growth process is discussed. MATERIALS AND METHODS Animals and Hormone Administration Adult 55- to 60-day-old female Sprague-Dawley rats weighing approximately 250 g were used. The animals were housed in a controlled environment at 200C on an illumination schedule of 12L: 12D. Standard pellet food and water were provided ad libitum. Ovariectomy was performed under light ether anesthesia 14 days before hormone treatment. The animal studies were approved by the Committee on Animal Care in Sweden and in accordance with the Guiding principles for the Care and Use of Research Animals promulgated by the Society for the study of Reproduction. Hormones Growth hormone (Genotropin) was a gift from Kabi/ Pharmacia (Stockholm, Sweden). Estradiol-173 (E2 ) and dexamethasone (DEX), purchased from Sigma Chemical Company (St. Louis, MO), were dissolved in 99.5% ethanol at a high concentration and then diluted with 0.9% NaCl to the proper concentration. The final ethanol concentration in the injections was less than 5%. Experiment I The animals received s.c. injections in the neck of the respective hormone in a volume of 100 pI1. DEX (250 txg) was given 3 h before an injection of E 2 (2.5 Vxg) or growth hormone (100 g), 24 h before animals were killed. Experiment II Five groups of animals were given s.c. injections of 2.5 ,g E2 (1 pig/100 g BW) and killed at various times after the injections as shown in Table 1. Preparation of Total Nucleic Acids Total nucleic acids (TNA) were prepared as described [13]. In brief, the tissues were homogenized and digested with proteinase K in an SDS-containing buffer; this was followed by extraction with phenol-chloroform as described by Durnam and Palmiter [14]. The concentration of total DNA in the TNA samples was measured fluorometrically at the wavelength 458 nm with Hoechst Dye 33258. Hybridization Probe The probe used for the thioredoxin mRNA determinations was derived from a clone of human thioredoxin cDNA
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THIOREDOXIN mRNA IN RAT UTERUS
1057
TABLE 1. Outline of treatment of animals in experiment II. Group II III IV V
Time(s) of injections(s) (h)
Time killed after injection (h)
Control group* 0 0 and 6 0, 6, 24, and 30 0, 6, 24, 30, 48, and 54
0 6 24 48 72
* Control rats received a volume of vehicle equivalent to treated rats, at 0 hours.
[15]. The thioredoxin cDNA (open reading frame coding for 105 amino acid residues) [16] subcloned into plasmid pACA [17] was isolated by EcoRI digestion and subsequently subcloned into the EcoRI site of the pGEM3Z vector. Two subclones with opposite orientations were selected and digested with Sma I. Sense and antisense RNA were obtained using T7 RNA polymerase. For measurements of specific mRNA, probes were synthesized in vitro and radiolabeled with [35 S]uridine triphosphate (UTP; Amersham, Buckinghamshire, UK). For Northern blot hybridization, the probe was instead labeled with [32 P]UTP. The in vitro synthesis of radioactive cRNA was performed essentially as described by Melton et al. [18], using reagents supplied from Promega Biotech (Madison, WI). Solution Hybridization Analysis of Thioredoxin mRNA Solution hybridization analysis was performed as described in Sahlin [19], with the following modification in order to be quantitative. The labeled hybrids protected from RNase digestion were precipitated by addition of 100 tpl 6 M trichloroacetic acid and collected on filters (Whatman GF/C; Whatman, Clifton, NJ). The radioactivity on the filters was monitored in a scintillation counter, and the results were compared with a standard curve of known amounts of in vitro-synthesized mRNA complementary to the probe used. Results are expressed as amol (10 18) mRNA/lug DNA in the TNA sample. Northern Blot Analysis of Thioredoxin mRNA A multiple-tissue Northern (MTN) blot containing poly(A)+ RNA prepared from specific tissues (Clontech, Palo Alto, CA) was prehybridized at 55°C in a solution containing 50% formamide, 5 mM sodium phosphate (pH 6.5), 5-strength SSC (single-strength SSC is 0.15 M NaCl/ 0.015 M sodium citrate), 0.1% SDS, 1 mM EDTA, 0.05% Ficoll, 0.05% polyvinylpyrrolidone, 0.05% BSA, and 200 pig/ml denatured salmon sperm DNA. After 4 h, a 32 P-labeled thioredoxin probe (1 x 106 cpm/ml prehybridization solution) was added, and hybridization was performed at 60°C for 40 h. The filters were washed in 0.1% SDS, 0.1strength SSC for 20 min at room temperature and 3 times for 20 min each at 65°C. The x-ray film (Amersham-hyperfilm MP) was exposed for 11 days at -70°C using intensifying screens. Statistics The results are presented as mean SEM. The evaluation was made by the Kruskal-Wallis test, and significance (p < 0.05) was determined by Dunn's test. Values with the same letter designation are not significantly different (p > 0.05).
FIG. 1. Analysis of the thioredoxin mRNA in rat tissues. A 32P-labeled human thioredoxin probe was hybridized to a rat MTN blot membrane. Each lane of the MTN blot contains 2 [Lg poly(A) + RNA from the respective tissue. Lane 1, heart; 2, brain; 3, spleen; 4, lung; 5, liver; 6, skeletal muscle; 7, kidney; and 8, testis. The sizes of the RNA markers are indicated in kilobases. The thioredoxin mRNA band is at approximately 0.6 kb.
RESULTS The human thioredoxin cRNA probe hybridized to an MTN blot from rat exhibited a single band in all the tissues analyzed except the skeletal muscle. In heart, brain, spleen, lung, liver, kidney, and testis a thioredoxin mRNA transcript of approximately 600 base pairs (bp) was present; it was very weak in the testis and most abundant in the liver, spleen, and kidney (Fig. 1). Ovariectomized rats treated with E 2 for 24 h, in which thioredoxin mRNA was analyzed with a solution hybridization method, showed a 4-fold increase in the uterus as compared to OVX controls (Fig. 2). The level in the E2treated group was not significantly different from those in the DEX- and GH-treated groups. When DEX was given in combination with E2, the level of thioredoxin mRNA
z 0
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z
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'o 0 I--Uvxc
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L
i
.
n
t 2*+ut2
UH+DeX
Treatment group FIG. 2. The levels of thioredoxin mRNA in the uteri of OVX rats after hormonal treatment. Ovxc, OVX control group. Values with different letter designations are significantly different (p < 0.05).
1058
SAHLIN ET AL.
6^ z0 C0) 0
if E
z a: E
C .X 0
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.o_ -
time (h) FIG. 3. Time-curve of thioredoxin mRNA induction in the uterus after E2 treatment. The thioredoxin mRNA level was determined from before treatment (Time 0) until 72 h after the first injection. Values with different letter designations are significantly different (p < 0.05).
increased 5-fold compared to the OVX control group; i.e., no synergism or antagonism was found between the hormones. The DEX+E 2 -treated group was also significantly different from the GH- and DEX-treated groups with respect to the thioredoxin mRNA level. The DEX-, GH-, and DEX+GH-treated groups did not differ from the OVX control group (Fig. 2). The time dependence of the increase in thioredoxin mRNA in the uterus after E2 treatment revealed a transient induction with a maximum after 48 h (Fig. 3). No response was seen 6 h after hormone exposure, and the level after 72 h was slightly lower than that at 24 h. In the liver, the level of thioredoxin mRNA was generally 10-fold higher than in the uterus when comparing the OVX control and time zero groups. Furthermore, no significant changes were seen after different hormonal treatments (Fig. 4) or during 72 h of E2 treatment by single injections (data not shown). This strongly indicates a specific effect of E2 on thioredoxin mRNA expression in the uterus. DISCUSSION The classic function of thioredoxin is as a hydrogen donor for the enzyme ribonucleotide reductase, which is es-
zZ 0
T T ~~T ......
3
...
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75 O
T
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z E C '~ 0
1*
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n=6 0I ovxc
*
n=6 E2
.
n6 DEX
I.
f=6 I GH
6
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=
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Treatment group FIG. 4. The levels of thioredoxin mRNA in the livers of OVX rats after hormonal treatment. Ovxc, OVX control group. No significant difference was found.
sential for DNA synthesis [20]. In addition, thioredoxin participates in the regulation of different metabolic systems via thiol redox control [1-4]. Such processes involve changes in the activity of different enzymes, receptors, or transcription factors via dithiol/disulfide interchange reactions [2, 21, 22]. Different mechanisms whereby thioredoxin could function during mitotic stimulation include functioning 1) as a general reductant of enzymes transporting electrons from NADPH; 2) as an antioxidant to balance the production of reactive oxygen intermediates involved in redox signaling mechanisms; or 3) as a physiological reductant of, e.g., growth factor receptors or enzymes with SH-groups critical for their activity. In the study reported here, we showed the presence of a single species of thioredoxin mRNA in rat tissues with the approximate size of 600 bp, which is in agreement with what has been reported previously [5, 6]. In the rat uterus, but not in the liver, the level of thioredoxin mRNA was strongly influenced by E2 treatment. DEX in combination with E2 did not exhibit any antagonistic effect as is the case during the E2 -induced insulin-like growth factor (IGF)-I mRNA expression in the uterus [19]. The absolute concentration of thioredoxin mRNA was higher in the liver than in the uterus, but the hepatic level was not affected by the hormonal treatment regimens tested in this study. GH was chosen for this study as a known inducer of IGF-I mRNA in the liver, in comparison to E2, a known inducer of IGF-I mRNA in the uterus [19]. GH did not affect thioredoxin mRNA levels in the liver, which implies that the induction of thioredoxin mRNA is not a general phenomenon after IGF-I mRNA induction. The biological functions of thioredoxin in the uterus during pregnancy or E2 exposure are likely to be the same as in other cells [1-4]. During these conditions there is increased mitogenic activity in the uterus and thus enhanced DNA, RNA, and protein synthesis, involving increased activity of enzymes and transcription factors, which in turn depend on increased thioredoxin activity; e.g., the transcription factor AP-1 (activator protein-1) has been shown to be under redox control [22, 23]. It has also been shown to be activated by estrogen treatment [24]. In a recent paper, Webb et al. [25] suggested a model for estrogen receptor (ER) regulation of AP-1 activity, according to which E2 stimulates promoter activity of AP-1. This may occur either in the "classical" way, in which the hormone-bound receptor binds as a dimer to the estrogen-responsive element (ERE) in the promoter region of a target gene, or via a DNA-binding domain-independent mechanism in which a single ER interacts directly with the Jun/Fos heterodimer. Alternatively, it may occur via a DNA-binding domain-dependent mechanism in which the ER interacts with an unknown target protein, activating a cascade that results in an increased transcription efficiency of Jun/Fos at their cognate AP-1 site [25]. We suggest that thioredoxin participates in this cascade, leading to AP-1 activation. Thioredoxin shown to be present in the human pregnant uterus [8, 9] could be induced by the increased serum E2 levels during pregnancy. In a separate study, we have observed that in the pregnant human cervix there is an increase in the thioredoxin mRNA level as compared to that in nonpregnant controls. There is also a positive correlation between the thioredoxin level in the cervix of nonpregnant women and the serum E2 level, whereas no correlation to the progesterone level has been found [26]. Cervical ripening is associated with an increase in collagen turnover, resulting in differently organized collagen fibrils. This
THIOREDOXIN mRNA IN RAT UTERUS
structural change is necessary for normal onset and progression of labor [27]. An AP-1 site exists in the promoter region of the collagenase gene [28]. Thus, estrogen could be involved in the slow remodeling of cervical collagen during pregnancy both directly and via stimulation of thioredoxin expression and its subsequent activation of AP-1. Further studies are needed to evaluate the function of thioredoxin in the uterus and cervix after estrogen exposure. ACKNOWLEDGMENT
13.
14. 15. 16.
We are grateful for excellent technical assistance from Britt Masironi.
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