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Endocrinology Vol. 147, No. 11 5034-5040
Copyright © 2006 by The Endocrine Society

Glucocorticoid Regulation of 24-Hour Oscillation in Interferon Receptor Gene Expression in Mouse Liver

Satoru Koyanagi, Hinako Suyama, Yukako Kuramoto, Noaya Matsunaga, Hiroshi Takane, Shinji Soeda, Hiroshi Shimeno, Shun Higuchi and Shigehiro Ohdo

Pharmaceutics (S.K., N.M., S.O.) and Clinical Pharmacokinetics (H.S., S.H.), Division of Clinical Pharmacy, Department of Medico-Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan; Department of Biochemistry (Y.K., S.S., H.S.), Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka 814-0180, Japan; and Department of Hospital Pharmacy (H.T.), Faculty of Medicine, Tottori University, Yonago 683-8504, Japan

Address all correspondence and requests for reprints to: Shigehiro Ohdo, Ph.D., Professor, Pharmaceutics, Division of Clinical Pharmacy, Department of Medico-Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, Kyushu University, 3-1-1 Maidashi, Higashi-Ku, Fukuoka 812-8582, Japan. E-mail: ohdo{at}phar.kyushu-u.ac.jp.


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
Although the antiviral effect of interferon (IFN) varies depending on 24-h oscillation in the expression of its specific receptor, the mechanism of oscillation remains to be clarified. Here we report that oscillation in the expression of the IFN receptor gene (IFN-{alpha}/ß R1) in mouse liver is caused by the endogenous rhythm of glucocorticoid secretion. Brief exposure of mouse hepatic cells (Hepa 1–6) to corticosterone (CORT) resulted in a significant decrease in mRNA levels of IFN-{alpha}/ß R1. The CORT-induced decrease in IFN-{alpha}/ß R1 mRNA levels was reversed by pretreating the cells with RU486, a glucocorticoid receptor antagonist. The mRNA levels of IFN-{alpha}/ß R1 gene in the liver of adrenalectomized mice were consistently increased throughout the day. However, a single administration of CORT to adrenalectomized mice significantly decreased the mRNA levels of IFN-{alpha}/ß R1 in the liver. Furthermore, the rhythmic phase of IFN-{alpha}/ß R1 expression was modulated after the alteration of rhythmicity in glucocorticoid secretion, which was induced by restricted daily feeding. As a consequence, under manipulation of the feeding schedule, 2'-5' oligoadenylate synthase activities, as an index of antiviral effect, in plasma and liver at 24 h after IFN-{alpha} injection also varied depending on the alteration of glucocorticoid secretion rhythm. These results suggest that the endogenous rhythm of glucocorticoid secretion is involved in the circadian regulation of IFN-{alpha}/ß R1 expression in mouse liver. Our findings also support the notion that monitoring the 24-h variation in IFN receptor function is useful for selecting the most appropriate time of day to administer IFN.


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
MAMMALIAN CIRCADIAN clocks, which generate various biological rhythms with a period length of about 24 h, are known to be located in the suprachiasmatic nucleus (SCN) of the hypothalamus. Recent molecular studies of the circadian biological clock system have revealed that oscillation in the transcription of specific clock genes plays a central role in the generation of 24-h rhythms (1, 2, 3, 4, 5, 6). The master clock located in the SCN follows a daily light/dark cycle and, in turn, synchronizes subsidiary oscillators in other brain regions and many peripheral tissues through neural and/or humonal signals (7, 8). These subsidiary oscillators coordinate a variety of biological processes, producing 24-h rhythms in physiology and behavior. The variations in biological function are also associated with the dosing time-dependent differences in the efficacy and/or toxicity of many drugs.

The daily rhythm of glucocorticoid secretion from the adrenal cortex is regulated by the hypothalamus-pituitary-adrenal axis, which in turn is controlled by the SCN (9). As glucocorticoids are involved in the regulation of a variety of physiological functions, such as energy metabolism and immunity function, 24-h changes in circulating glucocorticoid levels are thought to affect the efficacy of many drugs (10, 11).

Interferon (IFN), which belongs to a group of cytokines, has been widely used therapeutically as an antiviral and antitumor agent. In both human and experimental animals, it is well established that the pharmacological actions of IFN vary according to its administration time (12, 13, 14, 15, 16, 17). Although the dosing-time dependency of IFN-induced 2'-5' oligoadenylate synthase 2'-5' oligoadenylate synthase (OAS) activities, as an index of antiviral effect, is attributable to the 24-h variation in its specific receptor function (18), the underlying mechanism of the variation remains to be clarified. The biological effects of type I IFNs, such as IFN-{alpha} and -ß, are elicited by binding a specific receptor on the cell surface and are mediated through activation of the JAK-STAT signaling pathway (19). IFN-{alpha}/ß receptor is associated with two distinct component complexes, IFN-{alpha}/ß R1 and R2. It has been documented that the time-dependent variation in IFN receptor function is caused by rhythmic change in the expression of IFN-{alpha}/ß R1 (18).

In this study, we found that the brief exposure of mouse hepatic cells (Hepa 1–6) to corticosterone (CORT) significantly decreased mRNA levels of IFN-{alpha}/ß R1. Therefore, we designed this study to investigate the role of glucocorticoid in the circadian regulation of IFN-{alpha}/ß R1 expression in mouse liver. As the endogenous rhythm of glucocorticoid secretion was modulated by manipulation of the feeding schedule, we also explored how the modulation of glucocorticoid secretion rhythm influenced the antiviral effect of IFN-{alpha}.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Materials
IFN-{alpha} was purchased from PeproTech EC Ltd. (London, UK), dissolved in sterilized saline to a concentration of 2.0 MIU/ml, and stored frozen in aliquots at –20 C. CORT was purchased from Sigma Chemical (St. Louis, MO) and dissolved in sterilized saline containing 1% dimethyl sulfoxide for treatment.

Cell and animal experiments
Mouse hepatoma (Hepa 1–6) cells were purchased from ATCC (Manassas, VA) and cultured in DMEM (Invitrogen, Carlsbad, CA) supplemented with 10% fetal bovine serum. Male Institute of Cancer Research (ICR) mice (5 wk old) were purchased from Charles River Japan Inc. (Kanagawa, Japan). They were housed in a light-controlled room (lights on from 0700–1900 h) at a room temperature of 24 ± 1 C and humidity of 60 ± 10% with food and water available ad libitum or under a time-restricted feeding schedule (feeding time: 0900–1700 h). The animals were treated in accordance with the guidelines stipulated by the animal care and use committee of Kyushu University. To study the effect of CORT on IFN receptor expression in cultured cells, confluent cultures of Hepa 1–6 cells were incubated for 4 h in serum-starved (0.5% serum) medium containing various concentrations of CORT. As CORT binds to protein in the serum, we used low serum medium as the control vehicle. After incubation, total RNA was extracted using TRIzol reagent (Invitrogen). To investigate the role of glucocorticoid receptor in the action of CORT on IFN receptor expression, Hepa 1–6 cells were treated for 4 h with CORT (60 ng/ml) after pretreating with RU486 (10 µM), a glucocorticoid receptor antagonist, for 1 h. To examine the influence of the elimination of glucocorticoid secretion rhythm on the expression of IFN receptor mRNA, the temporal profile of mRNA levels for IFN receptor in the liver of adrenalectomized mice was investigated by RT-PCR method. The adrenals were removed via a dorsal approach using an aseptic technique under sodium pentobarbital anesthesia. The adrenalectomized mice were given 0.9% NaCl to drink during the experiment. Sham adrenalectomy was conducted by the same procedure to expose the adrenals without their removal. Liver was removed from the mice at one of the six times: 0900, 1300, 1700, 2100, 0100, or 0500 h. Total RNA from the tissue was prepared as described above. To explore the influence of CORT on the mRNA levels of IFN receptor in liver, adrenalectomized mice were given ip injections of 1.0 mg/kg CORT or vehicle. The dosage of CORT was selected based on a previous study (11). At 1 h after the injection of 1.0 mg/kg CORT to mice, the plasma concentration reached a similar daily peak to endogenous CORT levels. Total RNA from the liver was extracted at 0.5, 1.0, 2.0, 3.0, 4.0 and 6.0 h after CORT injection. To study the influence of altering glucocorticoid secretion rhythm on the expression of IFN receptor mRNA, the temporal profile of plasma CORT levels and hepatic IFN receptor expression was investigated under ad libitum feeding or a time-restricted feeding schedule. Blood samples were drawn by orbital sinus collection at one of the six times above. Immediately after blood collection, liver was removed from mice. Total RNA and membrane protein extractions from the liver were prepared at each time point. To examine 2'-5'OAS activities, as an index of the antiviral effect of IFN-{alpha}, mice were iv injected with 10.0 MIU/kg IFN-{alpha} or saline on two occasions: 0700 or 1900 h. Blood samples were drawn by cardiac puncture at 24 h after IFN-{alpha} injection and placed into polypropylene tubes containing 20 µl of EDTA (4%) solution. Immediately after blood collection, liver was perfused with 0.01 M PBS. The liver was quickly removed, rinsed with saline, and placed into ice-cold tubes.

Quantitative RT-PCR analysis
The synthesis and amplification of cDNA were carried using a superscript one-step RT-PCR system (Invitrogen). The sequence of mouse IFN {alpha}/ß receptor (IFN-{alpha}/ß R1) primers was as follows: 5'-AGAGTCAATGGGCAGTGTGA-3' and 5'-CGTTCCTGTGTAGACAGTATC-3' (GenBank accession number M89641). The sequence of ß-actin primers (internal control) was as follows: 5'-GAGGGAAATCGTGCGTGACAT-3' and 5'-ACATCTGCTGGAAGGTGGACA-3' (M12481). To quantify mRNAs, kinetic analysis of the amplified products, ensuring that signals were derived only from the exponential phase of amplifications, was performed in each sample as follows: after the first 24 cycles of amplification, an aliquot of 6 µg/ml was drawn for electrophoresis, and the tubes were submitted to one more cycle of PCR. This procedure was repeated until 28 cycles had been performed. The PCR products were run on 3% agarose gel. After the gel was stained with ethidium bromide, the density of each band was analyzed using Kodak ID image analysis software. The amplification efficiency of ß-actin and IFN-{alpha}/ß R1 genes was comparable. Therefore, the amplification products were collected and quantified at the 27th or 28th cycle. The ratio of the amplified target to the amplified internal control (calculated by dividing the value of each IFN-{alpha}/ß R1 by that of ß-actin) was compared among groups. In quantitative analysis, we set negative controls without inputting RNA or by omitting the RT step.

Determination of plasma CORT levels
Plasma samples were obtained after centrifugation at 3000 rpm for 3 min. These samples were heated at 56 C for 30 min to displace the CORT-binding protein. The plasma CORT concentration was determined by radioimmunosorbent assay (Corticosterone [125I] Assay System; Amersham Biosciences UK Ltd., Little Chalfont, Buckinghamshire, UK).

Immunoblot analysis
Membrane protein extraction from the liver was prepared as follows. The removed liver sections were washed with PBS, and homogenized with hypotonic lysis buffer (5 mM Tris-HCl, 2 mM EDTA, 5 µg/ml leupeptin, 10 µg/ml benzamide, 5 µg/ml trypsin inhibitor, pH 7.4). The homogenate was centrifuged to pellet insoluble fractions and nuclei. The supernatant was collected and centrifuged at 40,000 x g for 20 min, and the resulting pellet was washed and resuspended in lysis buffer (75 mM Tris-HCl, 12.5 mM MgCl2, 5 mM EDTA). Lysates containing 20 µg of total protein were resolved by SDS-PAGE and transferred to a polyvinylidene difluoride membrane. The membrane was reacted with antibodies against IFN-{alpha}/ß R1 chain (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Specific antigen-antibody complexes were visualized using horseradish peroxidase-conjugated secondary antibodies and Super Signal Chemiluminescent Substrate (Pierce Biotechnology, Inc., Rockford, IL). In this experiment, recombinant mouse IFN-{alpha}/ß R1 protein was used as a positive control. The recombinant protein was produced in pGEX expression vector after subcloning the fragment encoding the mouse IFN-{alpha}/ß R1 and purified by a glutathione agarose column.

Determination of 2'-5'OAS activities in plasma and liver
Plasma samples were obtained after centrifugation at 3000 rpm for 3 min and then stored at –20 C until assayed. Ice-cold liver was immediately homogenized with lysis buffer (10 mM HEPES-KOH/50 mM KCl/3 mM Mg(OAc)2/0.3 mM EDTA/10% glycerol/0.01% NaN3/0.5% Triton X-100/100 µM phenylmethylsulfonyl fluoride/7 mM 2-mercaptoethanol, pH 7.5). The supernatants, after centrifugation at 9000 g for 20 min at 4 C, were used as liver samples. The protein concentration in the liver homogenate sample was determined by Lowry’s method. Plasma and liver 2'-5'OAS activities were determined by RIA (2'-5'A kit; Eiken, Tokyo, Japan). The 2'-5'OAS activities in liver were expressed as 2'-5'oligoadenylate femtomoles per liver protein concentration.

Statistical analysis
ANOVA was applied for multiple comparison. Bonferroni’s test was used for multiple comparisons. A 5% level of probability was considered significant.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The effect of CORT on the expression of IFN-{alpha}/ß R1 gene in cultured mouse hepatic cells
In the first set of experiments, we investigated whether glucocorticoid could affect the mRNA expression of IFN receptors in cultured mouse hepatic cells. As CORT is the major glucocorticoid in rodents, we explored the influence of CORT on the mRNA levels of IFN receptors. Treatment of Hepa 1–6 cells with CORT for 4 h resulted in a significant decrease in IFN-{alpha}/ß R1 mRNA in a concentration-dependent manner; the most significant reduction was seen at a concentration of 60 ng/ml (Fig. 1AGo). Similar concentration-dependent reduction of IFN-{alpha}/ß R1 mRNA levels was also observed when cells were treated with dexamethasone, a potent glucocorticoid receptor agonist. However, treatment with both CORT and dexamethasone had little effect on the mRNA levels of IFN-{alpha}/ß R2 (data not shown). The inhibitory effect of CORT on the expression of IFN-{alpha}/ß R1 mRNA was reversed by pretreating the cells with RU486 (Fig. 1BGo). These results suggest that glucocorticoid has the ability to decrease IFN-{alpha}/ß R1 mRNA, probably via glucocorticoid receptor in mouse hepatic cells.


Figure 1
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  		FIG. 1. Effect of CORT on mRNA levels of IFN-{alpha}/ß R1 in cultured hepatic cells. A, Dose-dependent decrease in mRNA levels of IFN-{alpha}/ß R1 by CORT. Cultured Hepa 1–6 cells were treated for 4 h with CORT at the indicated concentrations. Total RNA was extracted and IFN-{alpha}/ß mRNA was analyzed by RT-PCR method. For intensity plots, the mean peak value of the untreated group was set at 1.0. ß-Actin mRNA was used as an internal control for transcripts. Each value represents the mean ± SEM (n = 3). **, P < 0.01; *, P < 0.05 compared with the untreated group. B, CORT-induced decrease in the mRNA levels of IFN-{alpha}/ß R1 in Hepa 1–6 cells was inhibited by RU486. Cultured Hepa 1–6 cells were exposed to 60 ng/ml CORT for 4 h after pretreatment or no pretreatment with RU486 (10 µM). The relative RNA level set the mean value of the untreated group to 1.0. ß-Actin mRNA was used as an internal control for transcripts. Each value is the mean ± SEM (n = 3). **, P < 0.01 when compared with the untreated group.

 
Adrenalectomy eliminated 24-h oscillation in IFN-{alpha}/ß R1 mRNA levels in the liver
We next investigated how the endogenous rhythm of glucocorticoid secretion affected the expression of IFN-{alpha}/ß R1 mRNA in mouse liver. As 24-h variation in plasma CORT levels is eliminated in adrenalectomized mice (10), we used these animals to investigate the influence of varying glucocorticoid levels on IFN-{alpha}/ß R1 expression in the liver. A significant time-dependent variation in IFN-{alpha}/ß R1 mRNA levels was observed in the liver of control (sham-operated) mice (P < 0.01, Fig. 2AGo). Higher levels of IFN-{alpha}/ß R1 mRNA were found from the late dark phase to the early light phase. By contrast, there was no significant time-dependent variation in IFN-{alpha}/ß R1 mRNA levels in the liver of adrenalectomized mice; the mRNA levels remained at peak levels in the control mice throughout the day (Fig. 2AGo). However, a single administration of exogenous CORT (1.0 mg/kg, ip) to adrenalectomized mice rapidly decreased the mRNA levels of IFN-{alpha}/ß R1 in liver. These results suggest that oscillation in IFN-{alpha}/ß R1 mRNA levels is caused by 24-h change in circulating glucocorticoid levels.


Figure 2
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  		FIG. 2. Influence of adrenalectomy on 24-h oscillation in mRNA levels of IFN-{alpha}/ß R1 in the liver. A, Temporal profiles of mRNA levels of IFN-{alpha}/ß R1 in the liver of sham-operated ({circ}) or adrenalectomized mice (bullet). For mRNA plot, the mean peak value of sham-operated mice was set at 100. ß-Actin mRNA was used as an internal control for transcripts. Each value represents the mean ± SE of four to six mice. **, P < 0.01, compared with the value for the sham-operated group at the corresponding times. B, Time course of the mRNA levels of IFN-{alpha}/ß R1 in liver after ip injection of 100 mg/kg CORT (bullet) or vehicle ({circ}) to adrenalectomized mice. Basal levels of each mRNA (at time point 0) were set at 1.0. ß-Actin mRNA was used as an internal control for transcripts. Each value represents the mean ± SE of three to four mice. **, P < 0.01; *, P < 0.05, compared with the value for the vehicle-treated group at the corresponding times.

 
Influence of feeding schedule on 24-h rhythms in the expression of IFN-{alpha}/ß R1 in liver
The act of feeding in mammals generates powerful cues for biological rhythms. For example, restricting the feeding time to an inactive period can reverse the rhythms of glucocorticoid secretion (20). Therefore, we explored whether manipulation of the feeding schedule can modulate rhythmicity in the expression of IFN-{alpha}/ß R1 genes in liver. Significant 24-h variation in plasma CORT levels was observed in control (sham-operated) mice in the ad libitum feeding group (P < 0.01; Fig. 3AGo). The levels of CORT increased from the late light phase to the early dark phase and decreased from the late dark phase to the early light phase. Under this feeding condition, mRNA levels of IFN-{alpha}/ß R1 in liver showed significant 24-h oscillation with higher levels from the late dark phase to the early light phase (P < 0.05; Fig. 3BGo). Similar 24-h oscillation was also found in protein levels of IFN-{alpha}/ß R1 in the liver of mice under the ad libitum feeding schedule (Fig. 3CGo). Time-restricted feeding had a marked influence on rhythmicity in both circulating CORT levels and the expression of IFN-{alpha}/ß R1. Higher levels of plasma CORT were observed around the early light phase, whereas lower levels were found from the late light phase to the early dark phase (Fig. 3AGo). Although both mRNA and protein levels of IFN-{alpha}/ß R1 in the liver showed obvious 24-h oscillations in the time-restricted feeding group, the rhythmic phase in the expression of IFN-{alpha}/ß R1 with manipulation of the feeding schedule was nearly antiphase to that under the ad libitum feeding schedule (Fig. 3Go, B and C).


Figure 3
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  		FIG. 3. Influence of time-restricted feeding on 24-h rhythms in plasma CORT level and hepatic IFN-{alpha}/ß R1 expression. A, Temporal profiles of CORT concentration in plasma of mice under ad libitum feeding ({circ}) or time-restricted feeding schedule (bullet). Each value represents the mean ± SE of six to eight mice. B, Temporal profiles of mRNA levels of IFN-{alpha}/ß R1 in the liver of mice with ad libitum feeding ({circ}) or the time-restricted feeding schedule (bullet). For mRNA plot, the mean peak value of mice under ad libitum feeding was set at 100. ß-Actin mRNA was used as an internal control for transcripts. Each value represents the mean ± SE of four to six mice. C, Temporal expression profiles of IFN-{alpha}/ß R1 protein in the liver of mice with ad libitum feeding or the time-restricted feeding schedule.

 
Influence of feeding schedule on dosing-time dependency of the antiviral effect of IFN-{alpha}
In the final set of experiments, we investigated the influence of the altered rhythm of IFN-{alpha}/ß R1 gene expression on the antiviral activity of IFN. To this end, the dosing-time dependency of IFN-{alpha}-induced 2'-5'OAS activities in liver and plasma was investigated under ad libitum feeding or time-restricted feeding. In both feeding groups, 2'-5'OAS activities in liver and plasma showed no significant differences between mice injected with saline at 0700 and 1900 h (Fig. 4Go). In the ad libitum feeding group, 2'-5'OAS activity in liver at 24 h after IFN-{alpha} injection at 0700 h increased significantly when compared with that after saline injection (P < 0.05; Fig. 4AGo), but 2'-5'OAS activity in liver at 24 h after IFN-{alpha} injection at 1900 h was not significantly different from that after saline injection. In the time-restricted feeding group, although 2'-5'OAS activity in liver at 24 h after IFN-{alpha} injection at 0700 or 1900 h increased when compared with that after saline injection (P < 0.05, respectively), 2'-5'OAS activity after IFN-{alpha} injection at 1900 h was higher than after injection at 0700 h.


Figure 4
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  		FIG. 4. Influence of time-restricted feeding on the dosing time-dependency of antiviral activity of IFN-{alpha}. A, 2'-5'OAS activities in liver at 24 h after IFN-{alpha} (10.0 MIU/kg, iv; closed column) or saline (open column) injection at 0700 or 1900 h. Each column represents the mean ± SE of eight to 10 mice. **, P < 0.01; *, P < 0.05 when compared with the saline group or two dosing times. B, 2'-5'OAS activities in plasma at 24 h after IFN-{alpha} (10.0 MIU/kg, iv, closed column) or saline (open column) injection at 0700 or 1900 h. Each column represents the mean ± SE of eight to 10 mice. **, P < 0.01; *, P < 0.05 when compared with the saline group or two dosing times.

 
In the ad libitum feeding group, 2'-5'OAS activity in plasma at 24 h after IFN-{alpha} injection was significantly higher for injection at 0700 h than for injection at 1900 h (P < 0.01; Fig. 4BGo). In the time-restricted feeding group, although 2'-5'OAS activity in plasma at 24 h after IFN-{alpha} injection at 0700 or 1900 h increased when compared with that after saline injection (P < 0.05, respectively), there was a significant dosing time-dependent difference in IFN-{alpha}-induced 2'-5'OAS activity (P < 0.05). Under this feeding schedule, 2'-5'OAS activity in plasma was significantly higher with injection at 1900 h than injection at 0700 h (P < 0.05). Taken together, these results suggest that the alteration of rhythmicity in IFN-{alpha}/ß R1 gene expression has a marked influence on the dosing-time dependency of 2'-5'OAS activity induced by IFN-{alpha}.


   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
Despite increasing evidence supporting a role of IFN-{alpha}/ß receptor in the pharmacological action of IFNs, little is known about the mechanism regulating time-dependent variation in the receptor’s function. In this study, we investigated the mechanism underlying the circadian control of IFN-{alpha}/ß R1 expression in mouse liver. Brief exposure of Hepa 1–6 cells to CORT significantly decreased the mRNA levels of IFN-{alpha}/ß R1. Furthermore, a single administration of CORT to adrenalectomized mice also resulted in a significant decrease in IFN-{alpha}/ß R1 mRNA levels in liver. These in vitro and in vivo data suggest that glucocorticoids directly act on hepatic cells and repress the expression of IFN-{alpha}/ß R1 mRNA.

Because the CORT-induced decrease in IFN-{alpha}/ß R1 mRNA levels was reversed by pretreating cells with a glucocorticoid receptor antagonist, glucocorticoids exert their action on gene expression through the activation of cytoplasmic glucocorticoid receptors that bind to glucocorticoid response elements (GREs). Several mechanisms by which glucocorticoids down-regulate transcription have been documented, and different types of negative GREs (nGREs) within the promoter region of target genes have also been identified (21, 22, 23, 24, 25, 26). However, the promoter architecture of the mouse IFN-{alpha}/ß R1 gene has been poorly characterized to date. Computer-aided analysis of the IFN-{alpha}/ß R1 promoter did not identify putative sequences resembling known nGRE, suggesting that there might be a novel type of nGREs in the IFN-{alpha}/ß R1 promoter. Alternatively, it is possible that glucocorticoids may activate glucocorticoid receptor complex and up-regulate the expression of some factors that repress the transcription of IFN-{alpha}/ß R1. Further studies are required to investigate the exact molecular mechanism of IFN-{alpha}/ß R1 repression by glucocorticoids.

Our present results are consistent with previously reported observations that hepatic IFN-{alpha}/ß R1 expression follows a 24-h oscillation (18). However, the 24-h rhythmicity in IFN-{alpha}/ß R1 mRNA levels was abolished by adrenalectomy, indicating that the endogenous rhythm of glucocorticoid secretion is responsible for the oscillation of IFN-{alpha}/ß R1 mRNA levels. Although the oscillation in the transcription of specific clock genes plays a central role in the generation of 24-h rhythms in various biological processes (27, 28), the rhythmic expression patterns of the clock genes, such as Clock, Bmal1, Per2, and Cry1, in the liver of the adrenalectomized mice did not differ significantly from those in the control (sham-operated) mice (see supplemental data 1, published on The Endocrine Society’s Journals Online web site at http://endo.endojournals.org). This fact suggests that adrenalectomy can eliminate the 24-h oscillation in IFN-{alpha}/ß mRNA levels in the liver without affecting the rhythmicity of molecular clock function. It is thus unlikely that the molecular components of the circadian clock are involved in the glucocorticoid-dependent regulation of IFN-{alpha}/ß R1 gene expression.

It has been documented that viral infection can induce the expression of IFN-{alpha}/ß receptors (29). This gene response to viral infection is thought to be attributable to cytokine action. For example, IFN-{gamma}, a major cytokine in the host defense to viral infection, has the ability to induce the expression of IFN-{alpha}/ß receptor mRNAs in hepatoma cells (30). However, the production of IFN-{gamma} from mouse lymphocytes is suppressed by CORT treatment (31). In fact, rhythmic phase of IFN-{gamma} expression in NK cells of rodents was nearly antiphase that of endogenous CORT secretion (32). Therefore, under a pathogenic condition, glucocorticoid may periodically suppress the production of a specific cytokine, thereby also contributing to the oscillation in the expression of IFN-{alpha}/ß R1 in liver.

Circadian rhythms in physiological functions are influenced by various environmental factors such as light, temperature, and social communication that vary cyclically in nature (29). Although light is the most powerful cue for physiological rhythms, the manipulation of feeding schedules can also modify the rhythms greatly (20). In fact, restricted daily feeding to nocturnally active mice had a marked influence on the rhythm in plasma CORT levels, accompanied by alteration of the rhythmicity in IFN-{alpha}/ß R1 expression. The alteration of IFN-{alpha}/ß R1 rhythm seemed to modulate the dosing-time dependency of IFN-{alpha}-induced 2'-5'OAS activity because the induction of 2'-5'OAS activities by IFN-{alpha} was also more pronounced in mice injected with the drug when IFN-{alpha}/ß R1 levels were increased. However, under time-restricted feeding conditions, there was a significant increase in 2'-5'OAS activities after IFN-{alpha} injection at the early light phase, even when the expression levels of IFN-{alpha}/ß R1 were low. Our previous data demonstrated that the dosing-time dependency of 2'-5'OAS activities induced by IFN-{alpha} is attributable not only to 24-h variation in IFN-{alpha}/ß receptor function, but also to its pharmacokinetics (15, 18). In fact, under the time-restricted feeding condition, higher IFN-{alpha} concentrations were still observed in mice injected with the drug when IFN-{alpha}/ß R1 levels were decreased (see supplemental data 2). Therefore, under the feeding condition, IFN-{alpha} concentrations in plasma after drug injection at the early light phase may be high enough to induce a significant increase in 2'-5'OAS activities.

The findings in this animal model suggest a mechanism underlying the time-dependent variation in IFN-{alpha}/ß R1 expression. The rhythmic secretion of glucocorticoids seems to induce 24-h oscillation in IFN-{alpha}/ß R1 expression in the mouse liver. Furthermore, manipulation of the feeding schedule appeared to modulate IFN-{alpha}-induced 2'-5'OAS activity by means of changing the endogenous rhythm of glucocorticoid secretion. As a consequence, manipulation of the feeding schedule and the choice of the most appropriate time of day for drug administration in relation to feeding time may help to achieve a rationale for the chronotherapeutics of IFN-{alpha} in certain experimental and clinical situations.


   Footnotes
 
This work was supported by a Grant-in-Aid for the Encouragement of Young Scientists from the Japan Society for the Promotion of Science (to S.K., 18790692), a Grant-in Aid for Scientific Research on Priority Areas "Cancer" (to S.O., 18014020), a Grant-in Aid for Scientific Research (B) (to S.O., 18390050), and a Grant-in Aid for Exploratory Research (to S.O., 18659042) from the Ministry of Education, Culture, Sport, Science and Technology.

Disclosure statement: The authors have nothing to disclose.

First Published Online August 17, 2006

Abbreviations: CORT, Corticosterone; GRE, glucocorticoid response element; IFN, interferon; nGRE, negative GRE; OAS, oligoadenylate synthase; SCN, suprachiasmatic nucleus.

Received March 31, 2006.

Accepted for publication August 8, 2006.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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