| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Department of Biochemistry (S.K., S.O., Y.K., H.S., S.S.), Faculty of Pharmaceutical Sciences, Fukuoka University, Fukuoka 814-0180, Japan; Pharmaceutics (K.U., S.O.), Division of Clinical Pharmacy, Department of Medico-Pharmaceutical Sciences, Faculty of Pharmaceutical Sciences, Kyushu University, Fukuoka 812-8582, Japan; and Division of Molecular Brain Science (H.O.), Department of Brain Sciences, Kobe University Graduate School of Medicine, Chuo-ku, Kobe 650-0017, 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 INTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
, and Bmal1 mRNA in HepG2 cells. Because the attenuation of clock gene oscillations was blocked by pretreating the cells with a Per1 antisense phosphothioate oligodeoxynucleotide, the extensive expression of Per1 induced by PSL may have resulted in the reduced amplitude of other clock gene oscillations. Continuous administration of PSL into mice constitutively increased the Per1 mRNA levels in liver and skeletal muscle, which seems to attenuate the oscillation in the expressions of Per2, Rev-erb, and Bmal1. However, a single daily administration of PSL at the time of day corresponding to acrophase of endogenous glucocorticoid levels had little effect on the rhythmic expression of clock genes. These results suggest a possible pharmacological action by PSL on the core circadian oscillation mechanism and indicate the possibility that the alteration of clock function induced by PSL can be avoided by optimizing the dosing schedule. |
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
and retinoid-related orphan receptor-, which drive a circadian oscillation in Bmal1 transcription (8, 9). In mammals, the master clock in the suprachiasmatic nuclei (SCN) of the anterior hypothalamus is entrained to a 24-h period by daily light-dark cycle. The master clock, in turn, synchronizes circadian oscillators in peripheral tissues through neural and/or humoral signals (10). Recently, it has been reported that retinoic acids, epidermal growth factor receptor, and glucocorticoid hormones function as humoral factors for resetting the peripheral oscillators (11, 12, 13). Among these substances, glucocorticoids have been considered to be particularly well suited for Zeitgeber for peripheral clocks. In fact, daily rhythm of glucocorticoid secretion is controlled by the hypothalamus-pituitary-adrenal axis, which in turn is regulated by the SCN (14, 15). Furthermore, a single administration of dexamethasone, a glucocorticoid receptor agonist, strongly induces phase shift of clock gene expression in peripheral tissues (13). These facts suggest that circadian secretion of glucocorticoid hormone acts to synchronize peripheral clocks with the central SCN pacemaker.
Circadian clock system is necessary to adapt endogenous physiological functions to daily variations in environmental conditions. Abnormality in circadian rhythms, such as the sleep-wake cycle and the timing of hormonal secretions, is implicated in various physiological and psychiatric disorders (16, 17). Although a large number of synthetic glucocorticoids are frequently used clinically as immunosuppressive or antiallergic agents, it remains poorly understood how the chronic treatment with synthetic glucocorticoid influences the circadian organization of mammalian clockwork.
In this study, we show that chronic treatment with synthetic glucocorticoid can alter the circadian clock function at molecular levels. Continuous administration of prednisolone (PSL), one of the most frequently used synthetic glucocorticoids for prolonged therapy, repressed the oscillation in the expression of clock genes in peripheral tissues of mice without changing the central SCN clockwork. The action of PSL on Per1 expression appears to have altered directly the molecular clock function in the peripheral cells. Because effectiveness and/or toxicity of many drugs vary according to their administration time, we explored the possibility that the alteration of clock function induced by glucocorticoid therapy could be overcome by optimizing the dosing schedule.
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
, treatment of HepG2 cells with PSL for 72 h resulted in an accumulation of Per1 mRNA in a concentration-dependent manner; the most significant accumulation was seen at a concentration of 0.5 µM. Dexamethasone also produced a similar concentration-dependent accumulation of Per1 mRNA (data not shown).
|
, upper panel). Treatment of the Per1 luciferase reporter vector-transfected HepG2 cells with 0.5 µM PSL for 48 h resulted in a 7.8-fold increase in the transcriptional activity (Fig. 1B, lower panel). This PSL-induced transactivation was thought to be dependent on the GRE, because a mutation of the AGAACACGATGTTCC sequence to AGAACACGATGGCTC decreased the transcriptional activity from 7.8-fold to 2.1-fold.
We also explored whether the PSL-induced accumulation of Per1 mRNA was caused by changing the stability of Per1 mRNA transcripts. HepG2 cells were incubated for 48 h in the presence or absence of 0.5 µM PSL. Then 10 µg/ml of actinomycin D was added to the media, and total RNA was extracted at selected time intervals. In vehicle-treated cells, the half-life of Per1 mRNA was approximately 2.98 h (Fig. 1C). However, the half-life was not obviously prolonged during exposure of the cells to PSL (3.07 h). Taken together, these results suggest that the PSL-induced accumulation of Per1 mRNA occurs at the transcriptional level rather than as a result of altered mRNA stability.
The chronic treatment with PSL also increased PER1 protein abundance in cultured hepatic cells (Fig. 2A, left panel). Because PER1 protein plays a critical role in mammalian circadian clock system through interaction with other clock gene products, we investigated how the PSL treatment influences the expression of other clock genes. Treatment of HepG2 cells with 0.5 µM PSL for 72 h substantially decreased the mRNA levels of Period2 (Per2), Cryptochrome1 (Cry1), D-site binding protein (Dbp), and Rev-erb, whereas it increased the levels of Bmal1 mRNA (Fig. 2A, left panel). Because mRNA levels of these clock genes were also modulated by transfecting the cells with PER1 expression constructs (Fig. 2A, right panel), we investigated whether the PSL-induced accumulation of PER1 resulted in alteration of mRNA levels of Per2, Cry1, Dbp, Rev-erb, and Bmal1 genes. As shown in Fig. 2B, the inductive effect of PSL on Per1 expression was blocked by pretreating the cells with the Per1 antisense phosphothioate oligodeoxynucleotide (ODN). The pretreatment with Per1 antisense ODNs also blocked the modulatory effect of PSL on mRNA expressions of Per2, Cry1, Dbp, Rev-erb, and Bmal1. These data suggest that PSL-induced accumulation of PER1 alters the mRNA levels of other clock gene.
|
, and Bmal1. To this end, luciferase reporter plasmids driven by promoters/enhancers of Per2, Rev-erb, or Bmal1 genes were constructed and transfected into HepG2 cells with PER1 expression vectors. As shown in Fig. 2C, the transcriptional activity of Per2 and Rev-erb was suppressed by cotransfection with PER1, whereas cotransfection of Bmal1 luciferase reporter with PER1 caused a 2.1-fold increase in the promoter activity. Because overexpression of PER1 had no significant effect on the half-lives of Per2, Rev-erb, and Bmal1 mRNAs (Fig. 2D), the PSL-induced accumulation of PER1 seems to modulate the expression of other clock genes at transcriptional level rather than as a result of their altered mRNA stabilities.
Influence of Chronic Treatment with PSL on the Rhythmic Expression of Clock Genes
We next examined how chronic treatment with PSL affects the rhythmicity of clock gene expression. The oscillation in the expression of clock genes in cultured hepatic cells was triggered by a 2-h treatment with 50% horse serum. Thereafter the cells were incubated in the presence or absence of 0.5 µM PSL. In control cells, which were incubated in the absence of PSL after serum treatment, mRNA levels of Per1, Per2, Rev-erb, and Bmal1 showed obvious circadian oscillations (Fig. 3, A and C). The expression patterns of clock genes were similar to those reported previously in rat-1 fibroblasts and vascular smooth muscle cells (11, 19). By contrast, chronic treatment of the serum-treated cells with 0.5 µM PSL decreased the amplitude of oscillations in clock gene expression. The levels of Per1 and Bmal1 mRNA were gradually increased during this treatment period, whereas the oscillations in the levels of Per2 and Rev-erb mRNAs were severely damped (Fig. 3, B and C). Similar decrease in the amplitude of clock gene oscillations was also observed in liver and skeletal muscle of mice infused continuously with PSL (10 µg/h, sc) using osmotic minipumps (Fig. 4). These results suggest that chronic treatment with PSL can affect the rhythmic expression of clock genes in peripheral tissues. Because the continuous administration of PSL into mice had little effect on the mRNA rhythms of Per1 and Per2 in the SCN (supplemental Fig. S1, published as supplemental data on The Endocrine Society’s Journals Online web sit at http://mend.endojournals.org), the effects of PSL on the clock genes’ expression in peripheral tissues are unlikely to be mediated via master clock function.
|
|
, and Bmal1 mRNAs in PER1 down-regulated cells. The expression of endogenous PER1 proteins was down-regulated by transfecting cells with Per1 antisense ODNs. The transfected cells were treated with 50% horse serum for 2 h and were subsequently incubated in the presence or absence of 0.5 µM PSL. In the absence of PSL, treatment of random ODN-transfected (control) cells with 50% horse serum resulted in significant time-dependent variations in the mRNA levels of Per1, Per2, Rev-erb, and Bmal1 (Fig, 3D). Although the time-dependent variations in clock gene expression were eliminated when the random ODN-transfected cells were incubated in the presence of PSL after the serum treatment, the inhibitory effects of PSL on the oscillations in the expression of clock genes were not observed in PER1-down-regulated cells (Fig. 3D). The clock gene expression patterns in PSL-treated PER1-down-regulated cells were similar to those observed in control cells. These results suggest that PSL-induced accumulation of PER1 proteins influences the rhythmic expression of other clock genes.
Changing the Dosing Schedule Minimizes the Disruptive Effect of PSL on Circadian Clock Function
Because continuous administration of PSL into mice modulated not only the circadian gene expression in peripheral tissues but also rhythmicity in locomotor activity and body temperature [Fig. 4 and supplemental Fig. S2 (published as supplemental data on The Endocrine Society’s Journals Online web sit at http://mend.endojournals.org)], we examined whether the alteration of clock function induced by PSL could be overcome by optimizing the dosing schedule. To this end, we subcutaneously administrated a single dose of PSL (5 mg/kg, sc), or vehicle [0.5% dimethylsulfoxide (DMSO) in saline] as a control, at Zeitgeber time (ZT)0 or ZT12 daily for 7 d. The expression rhythms of mRNAs of clock genes in liver were assessed on d 7 after initiation of the drug treatment. Although the repetitive administration of PSL at ZT0 substantially altered the mRNA rhythms of Per1, Per2, Rev-erb, and Bmal1 in liver, the administration of PSL at ZT12 had no significant effect on the rhythms of clock genes (Fig. 5).
|
). However, these overt rhythms were not significantly altered by the administration of PSL at ZT12. These results suggest that the chronic treatment with exogenous glucocorticoids does not always affect the molecular clock function in peripheral tissues. The alteration of clock function induced by PSL could be avoided by optimizing the dosing schedule.
|
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
, and Bmal1. Similar modulation of mRNA levels of those clock genes was also observed when the cells were transfected with PER1 expression constructs. The modulation of mRNA levels of clock gene appears to occur at transcriptional level rather than as a result of altered mRNA stability. The results of luciferase reporter gene analysis revealed that PSL directly acts on GRE in Per1 gene, thereby activating transcription of its mRNA. The transactivation of Per1 mRNA by PSL seems to result in the accumulation of its protein. Because the regulation of clock gene transcription is also regulated by other clock gene products (20, 21), the accumulation of PER1 protein is likely to influence the expression of other clock genes. This notion is also supported by the present findings that the modulation of mRNA levels of Per2, Cry1, Dbp, Rev-erb, and Bmal1 induced by PSL was nullified by pretreating the cells with Per1 antisense ODNs.
Chronic treatment with PSL also repressed the oscillation in the expression of Per1, Per2, Rev-erb, and Bmal1 in serum-shocked HepG2 cells. The circadian regulations of the transcription of Per2, Rev-erb, and Bmal1 genes are dependent on the functional clock-controlled elements. A noncanonical E box (CACGTT) in the Per2 gene, canonical E boxes (CACGTG) in the Rev-erb gene, and REV-ERB/retinoid-related orphan receptor-responsive elements in the Bmal1 gene are suggested to be responsible for rhythmic expression of their mRNAs (8, 9, 20, 21, 22, 23). CLOCK:BMAL1 heterodimers activate the transcription of Per2 and Rev-erb through an E box element, and the activation is inhibited by PER and CRY proteins (21, 22, 23). The REV-ERB protein also acts through retinoid-related orphan receptor-responsive elements to suppress the transcription of Bmal1, thereby regulating the rhythmic expression of its mRNA (8, 9). Therefore, PSL-induced accumulation of PER1 may result in repression of CLOCK/BMAL1-induced transactivation of Per2 and Rev-erb, which, in turn, leads to an increase in the levels of Bmal1 mRNA. This mechanistic interpretation may also be applicable to previous findings that glucocorticoids are involved in the circadian regulation of Rev-erb expression in mouse liver (24).
Consistent with its modulatory effect of PSL on the expression of clock genes in cultured hepatic cells, the continuous administration of PSL into mice prevented the oscillation in the expression of clock genes in the peripheral tissues. However, the treatment did not affect the mRNA rhythms of clock genes in the SCN. The cause may be that glucocorticoid receptor mRNAs do not accumulate in sufficient amounts to affect mouse SCN neurons (13). On a currently held model, mammalian circadian clock system is hierarchically organized: the master pacemaker in the SCN governs subsidiary oscillators in other brain regions and many peripheral tissues (25, 26). These subsidiary oscillators coordinate a variety of biological processes, producing overt rhythms in physiology and behavior (14, 15). Therefore, the direct action of PSL on molecular clock function of peripheral cells may lead to alteration of intrinsic rhythms of body temperature and locomotor activity.
Although the rhythmic expression of mRNA of clock genes in peripheral cells was changed in response to glucocorticoid stimuli, our present findings indicate that the peripheral oscillators would be less susceptible to exogenous glucocorticoid stimuli, when the glucocorticoid levels are endogenously increased. In nocturnally active rodents, the secretion of glucocorticoid hormones peaks from the late light phase to the early dark phase (14). The cyclic elevation of circulating glucocorticoid levels is thought to synchronize the oscillation in the expression of clock genes in peripheral cells. Therefore, the exogenous administration of PSL into mice at ZT0 may have impaired the endogenous nature of glucocorticoid rhythm and may have altered clock gene expression in their peripheral tissues. This may account for the fact that the disruptive effect of PSL on molecular clock function in peripheral cells can be lessened by choosing the optimal time for giving the dose.
Our results suggest that alteration of molecular clock function by PSL is the basis for adverse effects of glucocorticoid treatment on daily rhythms in physiology and behavior. The alteration of clock function seems to be elicited by the direct action of PSL on the Per1 gene. Altered homeostatic regulation, including the disturbance of the 24-h rhythms, is often observed in patients in chronic glucocorticoid therapy (27, 28, 29, 30). Our present finding may extend to understanding how chronic treatment with glucocorticoids modulates the molecular organization of circadian clockwork.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
Cell Culture and Transfection
HepG2 cells were maintained in DMEM supplemented with 10% fetal bovine serum. One day before transfection, the cells were seeded (1 x 105 per well) in six-well plates containing DMEM supplemented with 10% fetal bovine serum. Cells were transfected with 1.0 µg of PER1 expression constructs using LipofectAmine-Plus reagent (Invitrogen) according to the manufacturer’s instructions. To examine the influence of overexpression of Per1 on the mRNA levels of clock genes, total RNA from HepG2 cells was extracted at 72 h after transfection using TRIzol reagent (Invitrogen). Per1 antisense or random ODN was also transfected into the cells using LipofectAmine-Plus reagent. Twenty four hours after transfection, 0.5 µM PSL or vehicle (0.5% DMSO in saline) was directly added in a medium of Per1 antisense or random ODN-transfected cells. Total RNA was extracted after adding PSL for 48 h. To investigate the influence of PSL on the rhythmic expression of clock genes in PER1-down-regulated cells, Per1 antisense or random ODN was also transfected into the cells. Twenty four hours after transfection, Per1 antisense or random ODN-transfected cells were treated with 50% horse serum in medium for 2 h and subsequently incubated in the presence or absence of 0.5 µM PSL. After serum treatment, total RNA was extracted at 4-h intervals for 2 d.
Luciferase Reporter Gene Analysis
The promoter or enhancer regions of Per1 (–2207 to +24), Per2 (–118 to +10), and Bmal1 (–580 to +90) were amplified by PCR from mouse genomic DNA. pGL3-Rev-erb-luciferase reporter vectors (–3942 to +1) were kindly provided by Dr. Shimba (Nihon University, Chiba, Japan). Glucocorticoid responsive element (GRE) in the 5'-flanking region of mouse Per1 gene was mutated by using the QuikChange Site-Directed Mutagenesis kit (Stratagene, La Jolla, CA). HepG2 cells were transfected with 10 ng of reporter constructs and 1.0 (total) of expression vectors using LipofectAmine-Plus reagent. To correct for variations in transfection efficiency, 0.1 ng of pRL-TK vector (Promega Corp., Madison, WI) was cotransfected in all experiments. The total amount of DNA per well was adjusted by adding pcDNA 3.1 vector (Invitrogen). The ratio of firefly luciferase activity (expressed from reporter construct) to Renilla luciferase activity (expressed from pRL-TK) in each sample served as a measurement of normalized luciferase activity.
Quantitative RT-PCR Analysis
The cDNA of human or mouse Per1, Per2, Cry1, Dbp, Rev-erb, and Bmal1, and ß-actin were synthesized and amplified by using a superscript one-step RT-PCR system (Invitrogen). To evaluate the quantitative reliability of RT-PCR, a kinetic analysis of amplified products was done to ensure that signals were derived only from the exponential phase of amplifications (31). The exponential phase of ß-actin amplification in all experimental conditions occurred between the 26th and the 28th cycles, and the exponential phases of all target genes (clock genes) occurred between the 27th and the 30th cycles. The amplification efficiencies of ß-actin and clock gene were 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 Per, Cry, Dbp, Rev-erb, or Bmal1 by that of ß-actin) was compared among groups.
Determination of Half-Lives of Clock Gene’s mRNA
Actinomycin D (10 µg/ml) was added to HepG2 cells after 48-h incubation with or without 0.5 µM PSL. Cells were harvested at indicated times, and total RNA was extracted. RT-PCR analysis was performed as described above.
Western Blot Analysis
Nuclear fractions from HepG2 cells were prepared as described previously (31). The lysates containing 30 µg of total protein were loaded on SDS-PAGE, and the proteins were transferred to a polyvinylidene difluoride membrane. The membrane was reacted with antibodies against PER1 (Alpha Diagnostic, International, San Antonio, TX) or ß-ACTIN (Santa Cruz Biotechnology, Inc., Santa Cruz, CA). Specific antigen/antibody complexes were visualized by using horseradish peroxidase-conjugated secondary antibodies and Super Signal Chemiluminescent Substrate (Pierce Biotechnology, Inc., Rockford, IL).
Animals and Treatment
Male ICR mice (5 wk of age) were purchased from Charles River Japan, Inc. (Kanagawa, Japan). They were housed under a standardized light-dark cycle at room temperature of 24 ± 1 C and a humidity of 60 ± 10% with food and water ad libitum. The animals were adapted to the light-dark cycle for 2 wk before the experiments. During the dark period, a dim red light was used to aid treatment of the mice. An osmotic minipump (model 2001, ALZET; Alza Corp., Palo Alto, CA) was implanted under the skin of mice, and was used for continuous administration of PSL (10 µg/h) or vehicle (5% DMSO in saline). Total RNA from mouse tissues was extracted as follows: The brain, liver, and skeletal muscle were quickly removed at ZT2, ZT6, ZT10, ZT14, ZT18, and ZT22. Coronal brain slices (500 µm) were prepared using a rodent brain matrix (RBM-2000C; ASI Instruments, Inc., Warren, MI), and the SCN was punched out bilaterally from the brain slices. To obtain an adequate amount of RNA from the SCN, the SCN from three mice in each group were combined and extracted. Extraction of total RNA from the liver and skeletal muscle of each individual mouse was carried out separately by using the Trizol reagent.
Determination of Locomotor Activity
Locomotor activity was measured using a photobeam activity monitoring system (Muromachi Kikai, Co. Ltd., Tokyo, Japan), and activity count (number of movements) was recorded at 0.5-h intervals. The activity records were double plotted so that each day’s activity is shown both to the right and below that of the previous day. For visualization of locomotor activity rhythm, hourly activity counts were calculated using a moving average with a 4-h window.
Determination of Body Temperature
A lubricated thermocouple was inserted 1.5 cm into the rectum of mice, and the rectal temperature was determined using a digital thermometer (BAT-12; Bio Research Center, Tokyo, Japan).
Statistical Analysis
The significance of the 24-h variation in each parameter was tested by ANOVA. The statistical significance of differences among groups was analyzed by ANOVA and the Tukey multiple comparison test. A 5% level of probability was considered to be significant.
|
ACKNOWLEDGMENTS |
|---|
|
FOOTNOTES |
|---|
First Published Online November 3, 2005
Abbreviations: DMSO, Dimethylsulfoxide; GRE, glucocorticoid response element; ODN, oligodeoxynucleotide; PSL, prednisolone; SCN, suprachiasmatic nuclei; ZT, Zeitgeber time.
Received for publication April 22, 2005. Accepted for publication October 27, 2005.
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONRESULTSDISCUSSIONMATERIALS AND METHODSREFERENCES |
|---|
controls circadian transcription within the positive limb of the mammalian circadian oscillator. Cell 110:251–260[CrossRef][Medline] regulates circadian transcription of the mammalian core-clock Bmal1. Nat Struct Mol Biol 12:441–448[CrossRef][Medline] gene is a target of the circadian clock pacemaker. J Mol Endocrinol. 33:585–608 expression in liver. Endocrinology 141:3799–3806NURSA Molecule Pages Link:
This article has been cited by other articles:
 |
|
 |
|
  G. H. Son, S. Chung, H. K. Choe, H.-D. Kim, S.-M. Baik, H. Lee, H.-W. Lee, S. Choi, W. Sun, H. Kim, et al. Adrenal peripheral clock controls the autonomous circadian rhythm of glucocorticoid by causing rhythmic steroid production PNAS, December 30, 2008; 105(52): 20970 - 20975. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Burioka, S. Koyanagi, M. Endo, M. Takata, Y. Fukuoka, M. Miyata, K. Takeda, H. Chikumi, S. Ohdo, and E. Shimizu Clock gene dysfunction in patients with obstructive sleep apnoea syndrome Eur. Respir. J., July 1, 2008; 32(1): 105 - 112. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 N. Hayasaka, T. Yaita, T. Kuwaki, S. Honma, K.-i. Honma, T. Kudo, and S. Shibata Optimization of Dosing Schedule of Daily Inhalant Dexamethasone to Minimize Phase Shifting of Clock Gene Expression Rhythm in the Lungs of the Asthma Mouse Model Endocrinology, July 1, 2007; 148(7): 3316 - 3326. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 H. Okamura Suprachiasmatic Nucleus Clock Time in the Mammalian Circadian System Cold Spring Harb Symp Quant Biol, January 1, 2007; 72(0): 551 - 556. [Abstract] [PDF]
|
|
| |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
| Endocrinology | Endocrine Reviews | J. Clin. End. & Metab. |
| Molecular Endocrinology | Recent Prog. Horm. Res. | All Endocrine Journals |
)- or PSL (
)-treated cells. Data shown were confirmed in two independent vehicle-treated cells and three PSL-treated cells, respectively. Basal levels of each mRNA (at time point 0) were set at 1.0. The gray bars indicate the duration of serum treatment. D, Effect of down-regulation of PER1 protein on the PSL-induced modulation of circadian gene expression. Per1 antisense or random ODN-transfected cells were treated with 50% horse serum for 2 h and subsequently incubated in the presence or absence of 0.5 µM PSL. The mRNA levels of clock genes were determined at 28 h (
) and 40 h (
) after serum treatment. The levels of each mRNA were expressed as relative ratio to time point 0 (set at 1.0). Each value represents the mean ± SEM of three independent experiments. *, P < 0.05 compared with the value between the two times.
), ZT12 (