Departments of 1 Pediatrics, 2 Basic Nursing, and 4 Gynecology, Fukui Medical University, Fukui 910-1193; and 3 Institute for Virus Research, Kyoto University, Kyoto 606-8507, Japan
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ABSTRACT |
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We report here an
examination of the effect of thioredoxin (TRX) on the secretion of
growth hormone (GH) from rat anterior pituitary cells in vitro.
Treatment of rat pituitary cells with growth hormone-releasing factor
(GRF), but not GH, led to a significant increase in intracellular TRX
protein levels. GRF, recombinant human TRX (rhTRX), and a combination
thereof were all shown to induce immediate GH secretion from pituitary
cells, as evidenced by perifusion experiments. RhTRX, but not other
reducing agents such as -mercaptoethanol and
N-acetyl-L-cysteine, augmented GRF-stimulated and -unstimulated GH secretion from rat pituitary cells in a
dose-dependent manner. RhTRX did not significantly affect the GH mRNA
expression of pituitary cells stimulated in the presence or absence of
GRF. In addition, rhTRX-augmented GH secretion was not significantly affected by the presence of cycloheximide. Collectively, these findings
suggest that TRX is induced by stimulation with GRF and plays a
regulatory role in GH secretion from rat anterior pituitary cells by
enhancing the secretion of stored GH, rather than by the synthesis of GH.
redox; growth hormone-releasing factor; disulfide bonds
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INTRODUCTION |
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IT HAS RECENTLY BEEN SHOWN that reduction/oxidation (redox) reactions are intimately involved in the control of biological processes including the functional modulation of transcription factors (25, 32). In the case of the endocrine system, the cellular redox state appears to regulate the secretion and action of hormones. With regard to the hypothalamic-pituitary axis, a critical role for nitric oxide (NO), an endogenous redox modulator (34), in the regulation of growth hormone (GH) secretion has been proposed. We and others (7, 13) have recently reported that cultured rat pituitary cells tonically produce NO, which, in turn, blunts the growth hormone-releasing factor (GRF)-induced GH secretion through a guanosine 3',5'-cyclic monophosphate (cGMP)-independent mechanism.
An important constituent of the oxidant buffering system that controls the cellular redox state is thioredoxin (TRX), a 12-kDa protein with a redox-active disulfide/dithiol in the conserved active site sequence Cys-Gly-Pro-Cys (9, 25). This molecule has a variety of activities including serving as a hydrogen donor for various intracellular molecules (15, 24). Evidence has accumulated that suggests the presence of a control mechanism by the TRX system in certain endocrine systems (3, 8). For example, in the hypothalamic-pituitary-adrenal axis, TRX modulates cellular glucocorticoid responsiveness (6, 22). In the human ovary, adult T-cell leukemia-derived factor, the human form of TRX, exists and may participate in steroid hormone production (11). Recent immunohistological studies (28, 29) have demonstrated an intense level of staining for TRX in the pig anterior pituitary gland, supporting the contention that the TRX system may play a role in the regulation of GH secretion. However, to date, no data are available regarding the role of TRX in the GRF-GH axis.
In the present study, we report on an investigation of the effect of GRF on the synthesis of TRX in rat anterior pituitary cells and the regulatory role of TRX in GH secretion from these cells. Our findings present the first evidence that suggests that the TRX system, which is stimulated by GRF, acts as an enhancer of GH secretion in the rat anterior pituitary gland.
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MATERIALS AND METHODS |
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Pituitary cell dispersion.
Anterior pituitary glands were collected from male Sprague-Dawley rats,
aged 6-8 wk (Clea Japan, Tokyo, Japan). For 3 days before
decapitation, the animals were kept in our animal facilities at 24°C
on a 12:12-h light-dark cycle and received food and water ad libitum.
Primary pituitary cell cultures were prepared as described previously
(12, 14), with some modifications. Briefly, rat anterior
pituitaries were finely minced and incubated with 0.3% type I
collagenase (Sigma Chemical, St. Louis, MO) and 0.0009% DNase (Sigma
Chemical) in Hanks'-HEPES buffer containing 0.4% BSA at 37°C for
20-30 min. The pituitary suspension was triturated through pasteur
pipettes at 5-min intervals during the incubation.
Western blot analysis of GRF- or GH-induced TRX production.
Dispersed pituitary cells were cultured at 1.0-1.5 × 105
cells · ml1 · well
1 in
24-well culture plates (Corning, New York, NY) for 4-5 days. The
confluently grown cells were then reincubated with 1 ml of the fresh
culture medium/well for 24 h in the presence of 10
7
M human GRF-(1-44) (GRF; Peptide Institute, Osaka,
Japan) or 500 ng/ml rat GH (kindly provided by Dr. A. F. Parlow,
National Institute of Diabetes and Digestive and Kidney Diseases,
Bethesda, MD) during the last 0, 6, 12, and 24 h of the culture
periods. The TRX contents in the cells were then determined by Western blot analysis as described previously (31). Briefly, the
cultured cells were washed three times with ice-cold PBS and then
treated with a solubilizing buffer [0.5% octylphenoxyl
polyethoxyethanol (Nonidet P-40), 10 mM Tris · HCl, 150 mM
NaCl, 1 mM phenylmethylsulfonyl fluoride, 0.111 U/ml aprotinin, and
0.02% NaN3] on ice for 30 min. The resultant lysates were
centrifuged at 10,000 g for 10 min, and the supernatants
were used for SDS-PAGE. The concentrations of protein in the
supernatants were determined by the modified Lowry method (Bio-Rad
Laboratories, Hercules, CA). Equal amounts of proteins (8 or 10 µg)
were applied to each lane. After electrophoresis, proteins were
electrically transferred onto a nitrocellulose membrane (Millipore,
Bedford, MA). The membrane was blocked with 10% skim milk and 2% BSA
and then incubated with rabbit antiserum to murine TRX (1:2,000
dilution) at 4°C overnight, followed by horseradish peroxidase-linked
goat anti-rabbit immunoglobulins (1:100 dilution, according to the
manufacturer's instructions) (ENVISION+, Dako Japan, Kyoto, Japan).
Detection of the antigen-antibody complex was performed by enhanced
chemiluminescence (Amersham Pharmacia Biotech, Buckinghamshire, UK)
according to the manufacturer's instructions. Quantification of TRX
was performed by densitometric analysis with an imaging densitometer
(NIH image).
Effect of TRX, other reducing agents, and cycloheximide on GH
secretion.
Perifusion experiments were performed as previously described
(14), with minor modifications. Briefly, dispersed
pituitary cells were cultured with preswollen Cytodex microcarriers
type 3 (Amersham Pharmacia Biotech) in the culture medium at a ratio of
1.0 × 106 cells to 10 mg microcarriers in 3-cm
siliconized glass dishes. After 4-5 days of culture, the cells
were packed into Lucite columns with microcarriers and placed in a
37°C incubator. Three columns were perifused simultaneously with DMEM
at 0.5 ml/min and stimulated with DMEM containing GRF
(109 M) or rhTRX (100 µg/ml; Ajinomoto, Kawasaki,
Japan) or both for a 5-min period at 90-min intervals. The column
effluents were collected every 2 min by means of a fraction collector
and stored at
80°C for measurement of GH concentrations.
Measurement of GH concentrations. The concentrations of GH of the medium were measured by RIA, which was done in duplicate with a kit that was kindly provided by Dr. A. F. Parlow. RhTRX and other reducing agents at the concentrations used had no effect on the measurement of GH by RIA (data not shown).
Northern blot analysis of GH mRNA.
Dispersed pituitary cells were cultured at a density of
2.5-4.1 × 105 cells/well in 2 ml of the culture
medium in 6-well culture plates (Becton-Dickinson). After 4-5
days, the confluently grown cells were rinsed twice with DMEM and
incubated with 2 ml of DMEM containing rhTRX (100 µg/ml) and/or GRF
(109 M) for additional 4 h. At the end of the
incubation, cells were washed twice with ice-cold PBS. Total cellular
RNA was extracted with RNA isolation reagent (Isogen, Nippon Gene,
Tokyo, Japan). Three micrograms of RNA were separated on a 1% agarose
gel in 0.02 M 3-(N-morpholino)propanesulfonic acid buffer
and then transferred to a nylon membrane (Hybond N+, Amersham Pharmacia
Biotech). Hybridizations were performed using the rat GH cDNA probe
labeled with deoxy[
-32P]cytidine
5'-triphosphate, which was kindly provided by the Bioscience Research Institute of JCR Pharmaceuticals (Kobe, Japan). A final series
of washes was carried out at 2× saline-sodium citrate (SSC) buffer/0.1% SDS at room temperature, and 0.1 × SSC/0.1% SDS at 47°C. Quantification of GH mRNA was performed by densitometric analysis with the use of the NIH image.
Statistical analysis. All data are presented as means ± SE. Statistical comparisons were performed by one-way analysis of variance (ANOVA), with the use of the Bonferroni-Dunn test, and by the Student's t-test for the effect of cycloheximide and the effect of rhTRX on GH mRNA. P values <0.05 were considered statistically significant.
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RESULTS |
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Effect of GRF on intracellular TRX concentration.
The effect of GRF on intracellular TRX levels was first examined in rat
pituitary cells. Because our preliminary experiments showed that the
incubation of pituitary cells with GRF for 6 h was insufficient to
induce significant increases in TRX levels (data not shown), we
determined TRX levels after 12 and 24 h of incubation with GRF in
the following experiments. As shown in Fig.
1, A and B, the
cultured rat pituitary cells contained detectable amounts of TRX, and
stimulation of the cells with GRF significantly increased the
intracellular TRX protein levels. The mean TRX protein levels of cells
stimulated with GRF during the last 12 and 24 h of the culture
period were 2.6 and 2.3 times higher, respectively, than that observed
in the controls cultured without GRF throughout the culture period
(Fig. 1B). These results indicate that GRF caused an
increase in TRX protein levels within 12 h. In contrast, the TRX
protein levels were not altered by GH stimulation at any time examined
(Fig. 1, C and D). These results suggest that the GRF-induced increase in TRX is not due to an indirect effect via GH.
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Effect of TRX on GH secretion.
The possibility that TRX influenced GH secretion from rat pituitary
cells was then examined. In perifusion experiments, the GH secretion
significantly increased immediately after stimulation with
109 M GRF, 100 µg/ml rhTRX, or both (Fig.
2). The GH secretion reached maximum
within 5 min after the start of stimulation with each stimulus and
decreased rapidly after the cessation of the 5-min stimulation. The
maximum GH secretion generated by GRF plus rhTRX was higher than those
by GRF or rhTRX alone but was not statistically significant. The
stimulation was repeated 3 or 4 times after 90-min intervals in each
experiment, and the results were nearly the same (data not shown). The
cell viability at the end of the perifusion experiments was
90-93%, regardless of the stimuli (data not shown).
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Effect of cycloheximide on GH secretion.
Because the TRX-induced augmentation of GH secretion was observed
immediately after stimulation with rhTRX (Figs. 2 and 3), it is likely
that TRX shows its effect through the secretion of intracellularly
stored GH but through an augmentation in the de novo synthesis of GH.
To confirm this possibility, pituitary cells were stimulated with rhTRX
in the presence of cycloheximide. As shown in Fig.
4, cycloheximide had no significant
effect on the amounts of GH secreted by the cells treated with rhTRX
and/or GRF.
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Effect of TRX on GH mRNA expression.
As shown in Fig. 5, rhTRX had no effect
on GH mRNA levels in rat pituitary cells cultured with or without GRF,
even after 4 h. The data also suggest that rhTRX enhances the
secretion of stored GH but not the de novo synthesis of GH.
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Effects of other reducing agents on GH secretion.
Although rhTRX significantly augmented GH secretion,
-mercaptoethanol and N-acetyl-L-cysteine at
concentrations of 1 and 10 µM had no significant effect on GH
secretion (Fig. 6). The viability of
cells was not changed regardless of the treatments (data not shown).
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DISCUSSION |
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The physiological secretion of GH is generally thought to be regulated primarily by the two hypothalamic peptides GRF and somatostatin (5, 35), and redox modulation of disulfide bonds of hormones and receptors has been shown to influence GH secretion (4, 21, 36) as well as hormone-induced receptor activation (6, 8) and signal transduction (23). A redox-modulatory substance, NO, modulates the GRF-stimulated secretion of GH from rat pituitary cell cultures (13). TRX, another important redox-modulatory substance, has recently been identified in pig anterior pituitary gland by immunohistochemical analysis (28, 29). Immunoblotting analysis suggests that this is the case in calf and rat pituitary as well. This suggests that the TRX system is widely involved in the regulation of GH secretion from anterior pituitary gland. The data reported herein represent the first implication of the GRF-TRX-GH axis in rat pituitary gland.
The present findings show that stimulation with GRF increased TRX levels in rat anterior pituitary cells, and extrinsic rhTRX induced secretion of GH therefrom. Although the issue of whether GRF directly induces TRX production or indirectly induces it through induction/augmentation of other proteins is not clear, our results show that GH is not possibly the indirect inducer. The fact that TRX can be induced through a cAMP-dependent pathway (37), and that GRF is capable of activating a cAMP-dependent pathway in pituitary cells (1, 2, 4), suggests that GRF directly induces the synthesis of TRX protein in rat pituitary cells through a cAMP-dependent pathway.
Several possible explanations exist for the rhTRX-induced augmentation
of GH secretion from rat pituitary cells, including the induction of de
novo GH synthesis, augmentation of secretion of stored GH, or both. The
fact that stimulation with rhTRX immediately elicited GH secretion
(Fig. 2), that cycloheximide did not influence rhTRX-induced GH
secretion (Fig. 4), and that rhTRX did not appear to upregulate GH mRNA
expression (Fig. 5), suggests that rhTRX augments GH secretion by
increasing the secretion of stored GH but not through the induction of
the de novo synthesis of GH. GH is stored in pituitary secretory
granules in high concentrations in the form of intermolecular
disulfide-bonded oligomers (20), and the release of GH and
prolactin from isolated pituitary secretory granules is increased by
the presence of glutathione and other thiol-reducing agents, probably
through the disruption of disulfide bonds in the hormone oligomers
and/or granule membrane proteins (18, 19). It is likely
that TRX enters cells (33) and that its role in GH
secretion involves its strong reducing activity (10).
However, because two other reducing agents, -mercaptoethanol and
N-actyl-L-cysteine, failed to enhance GH
secretion in our study, it is possible that TRX may exert its role
through mechanisms other than simple reduction as well.
On the other hand, Lefrançois et al. (16) showed that reducing agents such as glutathione and dithiothreitol suppressed the coupling of GRF receptor with GRF. Other studies have also shown that the coupling of secretin or glucagon receptors with their ligands was decreased by thiol-reducing agents (17, 27, 30). Although such an inhibitory effect of TRX was not apparent in our experiments, it is possible that TRX, as well as other thiol-reducing physiological substances, has two opposing effects and regulates the GH secretion in a complex manner. Further analyses of the redox regulation of the GRF-GH axis may provide a better understanding of the characteristic pulsatile secretion of GH.
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ACKNOWLEDGEMENTS |
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We appreciate Dr. Manabu Inuzuka (Department of First Biochemistry, Fukui Medical University) for technical support. We also thank Dr. Yoshiki Yamamoto for providing the rat GH cDNA. RIA reagents were provided by Dr. A. F. Parlow through the National Hormone and Pituitary Program, National Institute of Diabetes and Digestive and Kidney Diseases. The recombinant human TRX was kindly provided by Aji-no-moto Central Laboratory, Japan.
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FOOTNOTES |
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Address for reprint requests and other correspondence: I. Hata, Dept. of Pediatrics, Fukui Medical University, Fukui 910-1193, Japan (E-mail: ikueh{at}fmsrsa.fukui-med.ac.jp).
The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
Received 23 October 2000; accepted in final form 16 March 2001.
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