The sympathetic nervous system modulates bone remodeling and mediates the expression of core clock genes in part through the β-adrenergic receptor (β-AR) in osteoblasts. In this study, we show that in MC3T3-E1 osteoblastic cells that isoproterenol (Iso), a non-selective β-AR agonist, upregulated the transcriptional factor Nfil3, and induced rhythmic mRNA expression of prostaglandin-endoperoxide synthase 2 (Ptgs2, also known as Cox2). The rhythmic effects of Iso on Ptgs2 expression were mediated by interplay between the Per2 and Bmal1 clock genes in osteoblasts. In addition, Ptgs2 was significantly decreased in bone after continuous Iso treatment. Overexpression of Nfil3 decreased Ptgs2 expression in MC3T3-E1 cells. Knockdown of Nfil3 upregulated the expression of Ptgs2 in MC3TC-E1 cells, indicating that Nfil3 negatively regulated Ptgs2 in osteoblasts. Furthermore, Iso acutely induced the expression Nfil3 and increased the binding of Nfil3 to the Ptgs2 promoter in MC3T3-E1 cells. These results suggest that Iso-mediated induction of Nfil3 in osteoblasts regulates the expression of Ptgs2 by driving the expression of circadian clock genes. These findings provide new evidence for a physiological role of circadian clockwork in bone metabolism.
Bone is a metabolically active organ that maintains continuous remodeling throughout life. Osteoblasts, osteocytes and osteoclasts are involved in bone remodeling processes in mammals, and these are regulated by endocrine, autocrine and paracrine signals (Raisz, 2005). Several studies have demonstrated that the sympathetic nervous system also regulates bone remodeling (Takeda et al., 2002; Elefteriou et al., 2005; Togari and Arai, 2008; Kondo and Togari, 2011) and mediates the expression of core clock genes, in part through the β-adrenergic receptor (β-AR), in osteoblasts (Fu et al., 2005).
The molecular machinery underlying circadian clocks in the suprachiasmatic nucleus (SCN; also known as the central pacemaker of the circadian clock) and peripheral tissues is composed of an autoregulatory transcriptional–translational feedback loop involving multiple clock genes, such as circadian locomoter output cycles protein kaput (Clock), brain and muscle Arnt-like protein-1 (Bmal1, also known as Arntl), period 1–3 (Per1–Per3) and cryptochrome 1 and 2 (Crys1 and Crys2), and their proteins, which are required for the generation of endogenous circadian oscillations (Ko and Takahashi, 2006). Circadian rhythms are ubiquitous in mammals, and are not only present in the SCN but also in various peripheral tissues (regarded as peripheral circadian oscillators) (Dibner et al., 2010). Indeed, an increasing number of investigations are focusing on circadian oscillators in bone. Sympathetic signaling and dexamethasone (Dex) have been shown to entrain bone circadian oscillators and the rhythmic expression of core clock genes in human osteoblasts (Komoto et al., 2012). This finding indicated that cells in bone share a similar molecular clock mechanism to that of the SCN. However, in spite of an increasing number of studies on circadian oscillators in osteoblasts, most have focused on the existence of circadian clock genes and their upstream stimulating factors. How the circadian oscillator in osteoblasts coordinates and regulates bone physiology in response to the sympathetic nervous system has yet to be elucidated.
In the present study, we determined that isoproterenol (Iso), a non-selective β-AR agonist, induced rhythmic mRNA expression of prostaglandin-endoperoxide synthase 2 [Ptgs2, also known as cyclooxygenase 2 (Cox2)] in MC3T3-E1 osteoblastic cells. We also demonstrated that nuclear factor interleukin-3 [Nfil3; also known as E4-binding protein 4 (E4BP4)], a negative regulator of circadian oscillation, played an important role in regulating Ptgs2, a most important regulator of bone metabolism.
β-AR signaling stimulated transcriptional rhythmicity in osteoblasts
To determine whether β-AR signaling in murine osteoblasts orchestrated rhythmic expression of the canonical clock gene Per2, MC3T3-E1 osteoblastic cells were treated with Iso at 1 µM for 2 h and Per2 expression was determined using quantitative real-time PCR (qRT-PCR) analysis at the indicated time points between 1 h and 56 h. Exposure to Iso acutely increased Per2 expression in a concentration-dependent manner (Fig. 1A) and induced rhythmic expression of Per2 mRNA and protein in MC3T3-E1 osteoblastic cells (the period of oscillation was 24.7 h) (Fig. 1B,C). Bmal1 is known to be indispensable in sustaining the transcriptional–translational feedback loop of Per2 (Kumaki et al., 2008). Therefore, we examined whether Bmal1 mediated Per2 expression in murine osteoblasts. Previous studies with continuous administration of Iso have revealed that chronic stimulation of β-AR by Iso reduced bone mass by increasing bone resorption and suppressing bone formation (Kondo and Togari, 2011). Therefore, we used a model of continuous stimulation of β-AR by administering Iso daily for 2 weeks with an osmotic pump, as described in the Materials and Methods. The results showed that Bmal1 and Per2 expression displayed a circadian pattern with opposing phases in bone (Fig. 2A). Furthermore, continuous treatment with Iso at 100 µg/g of body weight/day significantly increased Per2 expression at zeitgeber time (ZT)0, 12, 16 and 20 and decreased Bmal1 expression at ZT0 and ZT4 in bone (Fig. 2A). As shown in Fig. 2B, forced overexpression of the Bmal1–Clock complex significantly increased the expression of Per2 in MC3T3-E1 cells. We then attempted to elucidate the mechanisms regulating Per2 gene expression in MC3T3-E1 cells transfected with small interfering RNA (siRNA) for the knockdown of Bmal1 expression. Cells were transfected with siRNA for Bmal1, and the levels of Bmal1 were determined by real-time qRT-PCR. The results demonstrated that Bmal1 levels were significantly decreased in MC3T3-E1 cells transfected with Bmal1 siRNA 24 h after transfection (Fig. 2C). In addition, a significant decrease in Per2 expression was observed in MC3T3-E1 cells under these conditions (Fig. 2C). These finding indicate that circadian oscillators were impaired in osteoblasts treated with Bmal1 siRNA, which provides a prerequisite for the further detection of circadian-regulated genes in osteoblasts.
Ptgs2 is a circadian-regulated gene in osteoblasts
To understand the physiological function of the circadian clockwork on bone metabolism and to identify circadian-regulated genes affected by β-AR signaling in osteoblasts, we evaluated the expression of canonical core clock genes and Ptgs2 in bone after continuous Iso treatments by osmotic minipumps. As shown in Fig. 3A, we observed rhythmic expression of Ptgs2 over 24 h. In addition, continuous treatment of Iso decreased Ptgs2 expression at ZT0 and ZT12 and delayed the oscillation of Ptgs2 from ZT12 to ZT16 (Fig. 3A). Furthermore, Ptgs2 was significantly decreased at ZT0 by the continuous treatment with Iso at 25 and 100 µg/g of body weight/day in a dose-dependent manner (Fig. 3B), which indicated that Ptgs2 is one of circadian genes regulated by the sympathetic nervous system in bone. In order to determine whether β-AR signaling in osteoblasts could mediate the rhythmic expression of Ptgs2, cells were treated with Iso at 1 µM for 2 h and Ptgs2 expression were determined using real-time qRT-PCR analysis. Exposure to Iso entrained Ptgs2 with rhythmic expression in MC3T3-E1 osteoblastic cells (the period of oscillation was 28.5 h) (Fig. 4A). To further establish whether Bmal1-mediated transcription activation was necessary for circadian regulation of Ptgs2 mRNA in osteoblasts, we attempted to elucidate the regulation of Ptgs2 expression in MC3T3-E1 cells transfected with siRNA either against Bmal1 or Per2. Cells were transfected separately with siRNA against Bmal1 or Per2, followed by the determination of Ptgs2 levels using real-time qRT-PCR. On the one hand, a significant decrease was observed in Ptgs2 expression in Bmal1-knockdown MC3T3-E1 cells (Fig. 4B). On the other hand, a significant increase was observed in Ptgs2 expression in Per2-knockdown MC3T3-E1 cells (Fig. 4C). Taken together, these results suggest that Ptgs2 is one of the circadian genes regulated in response to β-AR signaling in osteoblasts.
Nfil3 negatively regulated Ptgs2 expression in MC3T3-E1 cells
Using a bioinformatics approach (http://www.cbrc.jp/research/db/TFSEARCH.html), we identified a putative consensus Nfil3/E4BP4 binding site in the Ptgs2 promoter from −723 to −712 bp upstream of the transcription start site. Nfil3 binds to a D-box element of the promoter region of target genes to repress transcription. Therefore, we expected Nfil3 to be one of the target molecules of β-AR signaling that regulated the circadian expression of Ptgs2 in osteoblasts. To investigate whether Nfil3 regulated Ptgs2 gene expression, we transduced MC3T3-E1 cells with either empty pcDNA3 (pcDNA3-Negative) or pcDNA3-Nfil3. Expression of Nfil3 was confirmed by western blotting (supplementary material Fig. S1). Our results showed that forced overexpression of Bmal1–Clock upregulated Ptgs2 gene expression in MC3T3-E1 cells, whereas forced overexpression of Nfil3 repressed it. Furthermore, overexpression of Nfil3 suppressed Bmal1–Clock-mediated upregulation of Ptgs2 expression in MC3T3-E1 cells (Fig. 5A). These results suggest that Nfil3 is involved in rhythmic expression of Ptgs2 in MC3T3-E1 osteoblastic cells. We then attempted to elucidate the relationship between Nfil3 and Ptgs2 in MC3T3-E1 cells. Based on our results (Hirai et al., 2014), transfecting MC3T3-E1 cells with siRNA against Nfil3, resulted in a significant decrease in Nfil3 by 24 h after transfection. Under these conditions, we observed a significant increase in Ptgs2 expression in MC3T3-E1 cells (Fig. 5B). These results indicate that Nfil3 negatively regulates Ptgs2 expression in MC3T3-E1 osteoblastic cells.
β-AR signaling regulated Nfil3 in MC3T3-E1 osteoblastic cells
We next characterized the expression of Nfil3 mRNA by β-AR signaling in bone. The systemic administration of Iso at 10 and 50 µg/g significantly increased the expression of Nfil3 mRNA in bone (Fig. 6A). Furthermore, in total RNA extracted from MC3T3-E1 osteoblastic cells following exposure to Iso for 1, 2, and 4 h, the expression of Nfil3 mRNA was significantly increased within 1 and 2 h after exposure to Iso. However, no significant alterations were observed in Nfil3 expression at 4 h following the exposure to Iso (Fig. 6B). As shown in Fig. 6C, exposure to Iso induced Nfil3 protein expression in MC3T3-E1 osteoblastic cells. In addition, pretreatment with the β-AR antagonist propranolol completely prevented Iso-induced Nfil3 expression in MC3T3-E1 osteoblastic cells, suggesting that upregulation of Nfil3 expression by Iso is mediated by β-AR signaling (Fig. 6D).
To determine whether Nfil3 directly regulated endogenous expression of the Ptgs2 gene in osteoblasts, we used the chromatin immunoprecipitation (ChIP) assay to confirm that Nfil3 was bound to the Ptgs2 promoter in MC3T3-E1 cells. Database analysis revealed a potential Nfil3 binding sites at −723 to −712 in the promoter, and at +2079 to +2090 (intron4), and +3344 to +3355 (intron5) and at +4147 to +4158 (intron7) in the Ptgs2 gene. ChIP experiments on MC3T3-E1 cells were conducted using rabbit polyclonal anti-sera, followed by agarose gel electrophoresis. These assays revealed basal occupancy of Nfil3 to the element located at −723 to −712 in the Ptgs2 promoter (Fig. 7A,B). Moreover, Nfil3 binding to the D-box-containing Ptgs2 promoter region was increased in MC3T3-E1 cells after the Iso treatments (Fig. 7C). Taken together, these results indicate that the expression of Nfil3 induced by β-AR signaling directly bound to the Ptgs2 promoter and inhibited Ptgs2 expression in osteoblasts.
In the present study, we attempted to unravel the physiological implications of circadian oscillators in response to sympathetic nervous system activation in osteoblasts. Our results suggest that β-AR signaling increases the expression of Nfil3 and, consequently, its binding to the Ptgs2 promoter, which in turn inhibits Ptgs2 expression in osteoblasts (supplementary material Fig. S2).
Recent studies conducted in our laboratory have shown that treatments with Iso or Dex induce circadian expression of the clock genes Per1, Per2, Per3 and Bmal1 in human osteoblasts. On the one hand, Iso induced oscillations in the osteoblast-related gene Col1a1, but not in ALP or osteocalcin (Komoto et al., 2012). On the other hand, we did not observe any significant effects of Iso in osteoclasts, which indicates that Dex rather than Iso signaling governs the transcriptional rhythmicity of the molecular clock in osteoclasts (Fujihara et al., 2014). These findings suggest that the Iso-mediated effects on Per2 expression in bone were reflected in osteoblasts. The results of the present study supply further evidence that Iso induces rhythmic mRNA expression of the clock gene Per2 in MC3T3-E1 osteoblastic cells (Fig. 1). Additionally, Per2 was significantly downregulated by the Bmal1 siRNA, which implies that the molecular machines underlying circadian clocks in osteoblasts, as well as in other cell types, are composed of transcriptional–translational feedback loops involving clock genes that are mediated by E-box elements located in their promoter regions (Sato et al., 2006). However, we cannot deny the possibility that they might be under the regulation of clock-regulated genes, rather than core clock genes.
Ptgs2 is an important rate-limiting enzyme that determines the production of prostaglandin E2 (PGE2), which mediates inflammatory responses, in addition to being one of the most important regulators of bone metabolism (Blackwell et al., 2010). Osteoblasts and osteocytes increase the expression of Ptgs2 and release PGE2 in response to fluid flow stress (Wadhwa et al., 2002; Cherian et al., 2003). In addition, Ptgs2 regulates mesenchymal cell differentiation into the osteoblast lineage (Zhang et al., 2002) and osteoclast maturation (Blackwell et al., 2010). Rhythmic expression of Ptgs2 mRNA levels was confirmed by the Cosinor method (Fig. 4A), indicating that circadian oscillators in osteoblasts might modulate diverse physiological processes by regulating Ptgs2, which in turn might regulate PGE2 secretion and bone remodeling. The bioinformatics approach used in our studies revealed that the Ptgs2 gene contains E-box and D-box sequences within the promoter, suggesting that Ptgs2 is a circadian gene that is regulated by circadian oscillators in osteoblasts. Furthermore, downregulation of Bmal1 significantly decreased the expression of Ptgs2 mRNA (Fig. 4B), which illustrated that rhythmic expression of Ptgs2 is generated by its own transcriptional–translational feedback loop. This feedback loop might involve a set of clock genes that are regulated by the Bmal1–Clock heterodimer in MC3T3-E1 osteoblastic cells. Previous reports have shown that Per2 genes have a cyclic AMP response element as well as D- and E-box-binding elements in their promoter (Yoo et al., 2005; Yagita and Okamura, 2000; Ohno et al., 2007). In the present study, acute Iso treatment increased Per2 expression in MC3T3-E1 cells (Fig. 1A), whereas chronic Iso infusions increased Per2 expression and delayed the oscillation of Ptgs2 in bone (Fig. 2A; Fig. 3A). In addition, knockdown of Per2, which repressed Bmal1 transcription, significantly increased Ptgs2 expression (Fig. 4C). Therefore, Iso-mediated upregulation of Per2 might play a role in the mechanism of Iso-mediated regulation of Ptgs2 in osteobasts.
The activity of the sympathetic nervous system displays circadian rhythmicity (Herzog, 2007). In addition, β-AR signaling is involved in mediating circadian rhythms in bone metabolism (Fu, et al., 2005), in negatively regulate bone mass by inhibiting osteoblast proliferation (Takeda et al., 2002; Elefteriou et al., 2005) and in activating osteoclast activity (Togari and Arai, 2008; Kondo and Togari, 2011). Consistent with previous studies (Komoto et al., 2012), β-AR signaling mediated clock genes such as Per2 and Bmal1 in osteoblasts (Fig. 2). The bZIP transcription factor Nfil3/E4BP4 is a mammalian homolog of vrille, which functions as a key negative component of the Drosophila circadian clock (Blau and Young, 1999; Cyran et al., 2003, Glossop et al., 2003) and is believed to be involved in the mammalian circadian clock (Mitsui et al., 2001; Ueda et al., 2005; Akashi et al., 2006). Additionally, Nfil3 plays an essential role in natural killer (NK) cell development and IgE class switching (Gascoyne et al., 2009; Kamizono et al., 2009; Kashiwada et al., 2010). In the present study, we showed that Nfil3 was upregulated by β-AR signaling in osteoblasts. The detailed mechanisms by which Nfil3 is upregulated in osteoblasts remain to be established. Previous reports have shown that the expression of Nfil3 is upregulated by elevated intracellular Ca2+ concentration and by activation of cAMP-response-element-binding protein (MacGillavry et al., 2009; Nishimura and Tanaka, 2001). Considering that Nfil3 was upregulated in MC3T3-E1 cells exposed to Iso, it is possible that cAMP/PKA pathways might trigger upregulation of Nfil3 in MC3T3-E1 cells. However, given that the expression of Nfil3 mRNA is not sustained beyond 4 h, it would suggest that additional posttranscriptional mechanisms such as mRNA destabilization play a later role. Physiologically, it appeared that Nfil3 exerted a negative regulatory effect on Ptgs2 expression in osteoblasts because Ptgs2 transcripts were downregulated in Nfil3-overexpressed MC3T3-E1 cells and upregulated in Nfil3-silenced MC3T3-E1 cells (Fig. 5).
Our results reveal that the effects of Iso on Ptgs2 expression were exerted by a complex counter regulatory mechanism. On the one hand, the rhythmic effects of Iso on Ptgs2 expression that were described in Fig. 4 were mediated by an interplay between the Per2 and Bmal1 clock proteins at the E-box of the Ptgs2 promoter (supplementary material Fig. S2). On the other hand, Iso-mediated chronic activation β-AR signaling increased the expression of Nfil3 (RT-PCR) and its binding to D-box elements in the Ptgs2 promoter (ChIP assay), which in turn inhibited the expression of Ptgs2 (supplementary material Fig. S2). These results provide a molecular mechanism for the control of bone metabolism by circadian rhythms.
In conclusion, our results suggest that β-AR signaling in osteoblasts regulate Ptgs2 through upregulation of Nfil3, a negative regulator of circadian oscillations, and thus provide new evidence for the physiological importance of the circadian clockwork in bone metabolism.
MATERIALS AND METHODS
Male eight-week-old C57BL/6J mice (Japan SLC, Hamamatsu, Japan) were randomized by weight, assigned to groups, and acclimated to their cages for 1 week prior to the experiment. They were treated in accordance with the Guidelines for Animal Experiments at the School of Dentistry, Aichi-Gakuin University. Food and water were available ad libitum. Animals were housed together in automatically controlled conditions of temperature (23±1°C) and humidity (50±10%) under a 12-hour-light–12-hour-dark cycle or under constant dark conditions. Zeitgeber time (ZT) 0 under the light–dark cycle was designated as lights on and ZT12 as lights off.
Mice were administered isoproterenol (Sigma-Aldrich) at 100 µg/g body weight daily for 2 weeks or saline as a vehicle control by osmotic minipumps (Alzet model 2002; Durect, Cupertino, CA) implanted into the subcutaneous tissue in the back over a period of 14 days, according to the manufacturer's instructions. On the last day of the experiment, all mice were killed by cervical dislocation under ether anesthesia, and the femora and tibiae were collected (Fujihara et al., 2014). Bone tissue samples were dissected and kept at −80°C until assayed for total RNA.
MC3T3-E1 cell cultures
MC3T3-E1 cells were purchased from the RIKEN Cell Bank. MC3T3-E1 cells were cultured in α-MEM (Life Technologies) containing 10% FBS and 1% penicillin-streptomycin at 37°C under a 5% CO2 atmosphere. To induce differentiation, the culture medium was replaced with α-MEM containing 50 µg/ml ascorbic acid and 5 mM β-glycerophosphate. The culture medium was changed every 2–3 days.
MC3T3-E1 cells were grown in α-MEM supplemented with 10% FBS and 1% penicillin-streptomycin to ∼70% confluency, followed by transient transfection with either small interfering RNA (siRNA) targeting genes or non-silencing RNA diluted in Opti-MEM using Lipofectamine RNAiMAX (Life Technologies) according to the manufacturer's protocol. Silencer Select siRNAs were used (Ambion/Applied Biosystems). Bmal1, Per2, Nfil3 and non-silencing siRNAs were used at final concentrations of 10 nM. The medium was then replaced with fresh medium. Cells were harvested for total RNA extraction at the indicated time points.
Construction of expression plasmids and transfection
The entire coding sequence of mouse Nfil3 cDNA was amplified by KOD-plus Neo DNA polymerase (Toyobo, Japan) and inserted into the mammalian expression vector pcDNA3. The primers used were as follows: forward 5′-CGCGGATCCTGGGGAAGTGATTTGTCTCC-3′ and reverse 5′-CCGCTCGAGTCCTCCTCCATGCATAGCTC-3′. Amplified DNA was cloned into the pcDNA3 vector at the BamHI and XhoI restriction enzyme sites (underlined). The correct sequences of the subcloned cDNA fragment were confirmed by complete nucleotide sequencing. The other plasmids used in this study were obtained from Addgene. mClock and mBmal1 cDNA were cloned into pcDNA4 to produce pCKPC4, which expresses hexahistidine- and Flag-tagged mouse CLOCK (pcDNA-Clock), and also into pcDNA3 to produce pBMPC3, which expresses hexahistidine-tagged mouse BMAL1 (pcDNA-Bmal1). MC3T3-E1 cells were plated at a density of 1.0×105 cells/cm2. Cells were transfected after 24 h with pcDNA-Nfil3, pcDNA-Bmal1, pcDNA-Clock or the empty vector using FugeneHD reagent (Promega) according to the manufacturer's instructions.
RNA extraction and quantitative RT-PCR
As an in vivo experiment, total RNA was extracted from the femur (cancellous bone). Total RNA was isolated with an RNeasy Mini Kit (Qiagen) according to the manufacturer's protocol. A total of 1 µg RNA was reverse transcribed into cDNA using the QuantiTect Reverse Transcription Kit according to the protocol of the manufacturer (Qiagen). Gene expression was analyzed with the Step-One-Plus real-time PCR system with Step One Software v2.0 (Applied Biosystems). Reactions were performed in 20-µl volumes using a QuantiTect SYBR Green PCR Kit (Qiagen). Cycling conditions were 50°C for 2 min, 95°C for 10 min, followed by 40 cycles of 95°C for 15 s and 60°C for 1 min. The relative quantification of each mRNA was performed using the comparative quantity (copies) method, creating standard curves. Relative standard curves were created with serially diluted cDNA samples that were reverse transcribed from RNA samples. This concentration range of standard curve samples was determined to be well within the detection level and sensitivity of the qRT-PCR assay. The relative quantity for each sample was normalized to the average level of the constitutively expressed housekeeping gene Gapdh (Komoto et al., 2012). The following primers were used: Gapdh, forward 5′-TGGAGAAACCTGCCAAGTATG-3′, reverse 5′-GGAGACAACCTGGTCCTCAG-3′; Nfil3, forward 5′-CAGTGCAGGTGACGAACATT-3′, reverse 5′-TTCCACCACACCTGTTTTGA-3′; Ptgs2, forward 5′-TGCCTGGTCTGATGATGTATG-3′, reverse 5′-GGGGTGCCAGTGATAGAGTG-3′; and Per2, forward 5′-ATGCTCGCCATCCACAAGA-3′, reverse 5′-GCGGAATCGAATGGGAGAAT-3′.
Western blotting analysis
Cells were washed with PBS and collected in 20 mM Tris-HCl buffer (pH 7.4) containing 1 mM EDTA, 1 mM EGTA, and the previously described inhibitors of phosphatases and proteases (Hirai et al., 2011). Cell homogenates were mixed at a volume ratio of 4∶1 with 10 mM Tris-HCl buffer (pH 6.8) containing 10% glycerol, 2% sodium dodecyl sulfate (SDS), 0.01% Bromophenol Blue and 5% mercaptoethanol, followed by boiling at 100°C for 5 min, as previously described (Hirai et al., 2006). Each aliquot of protein was subjected to electrophoresis on a polyacrylamide gel containing 0.1% SDS at a constant current of 10 mA/plate at room temperature and was subsequently transferred onto a polyvinylidene fluoride membrane. The membranes were subjected to blocking with 5% low-fat milk dissolved in TBST (20 mM Tris-HCl pH 7.5 containing 137 mM NaCl and 0.05% Tween-20), followed by a reaction with antibodies against Nfil3 and Per2 (Santa Cruz Biotechnology) adequately diluted in 1% low-fat milk in TBST. Finally, proteins were reacted with the anti-rabbit-IgG antibody conjugated to horseradish peroxidase and detected with the aid of ECL detection reagents.
Chromatin immunoprecipitation assay
The ChIP assay was performed using ChIP-IT express (Active Motif) according to the manufacturer's instructions. Cell extracts were immunoprecipitated with polyclonal rabbit anti-Nfil3 (Santa Cruz). Antibody specificity was assayed with normal rabbit IgG (Santa Cruz Biotechnology). Purified DNA fragments were subjected to PCR, assessing for the Ptgs2 promoter region −665 to −530, using the following primers: 5′-CGATGAAGTGGAGCTCAGCA-3′ and 5′-TCCTCTGTCAGGGTCCACAT-3′. PCR products were also analyzed on 2.5% agarose gels.
All data are expressed as the mean±s.e.m. Differences between groups were tested for statistical significance using the unpaired two-tailed Student's t-test. P<0.05 was considered to indicate significance. The circadian rhythmicity in gene expression was determined by the single Cosinor method using Timing Series Analysis Single Cosinor 6.3 Software (Expert Soft Tech.). The period was determined by using the chronobiometric ellipse test (Komoto et al., 2012).
Study design was undertaken by T. H. Study conduct was undertaken by T. H. and T. K. Data interpretation was undertaken by T. H. and T. K. Manuscript preparation was undertaken by T. H. Drafting of the manuscript was undertaken by T. H. Manuscript revision was undertaken by T. H. Approving final version of manuscript was undertaken by T. H., T. K. and A. T.; T. H. and A. T. take responsibility for integrity of the data analysis.
This work was supported in part by Grants-in-Aid for Scientific Research [grant number 24790291 to T.H.] from the Ministry of Education, Culture, Sports, Science and Technology, Japan.
The authors declare no competing interests.