Background & Aims
The circadian clock drives daily rhythms in behavior and physiology. A recent study suggests that intestinal permeability is also under control of the circadian clock. However, the precise mechanisms remain largely unknown. Because intestinal permeability depends on tight junction (TJ) that regulates the epithelial paracellular pathway, this study investigated whether the circadian clock regulates the expression levels of TJ proteins in the intestine.
The expression levels of TJ proteins in the large intestinal epithelium and colonic permeability were analyzed every 4, 6, or 12 hours between wild-type mice and mice with a mutation of a key clock gene Period2 (Per2; mPer2m/m). In addition, the susceptibility to dextran sodium sulfate (DSS)-induced colitis was compared between wild-type mice and mPer2m/m mice.
The mRNA and protein expression levels of Occludin and Claudin-1 exhibited daily variations in the colonic epithelium in wild-type mice, whereas they were constitutively high in mPer2m/m mice. Colonic permeability in wild-type mice exhibited daily variations, which was inversely associated with the expression levels of Occludin and Claudin-1 proteins, whereas it was constitutively low in mPer2m/m mice. mPer2m/m mice were more resistant to the colonic injury induced by DSS than wild-type mice.
Citation: Oh-oka K, Kono H, Ishimaru K, Miyake K, Kubota T, Ogawa H, et al. (2014) Expressions of Tight Junction Proteins Occludin and Claudin-1 Are under the Circadian Control in the Mouse Large Intestine: Implications in Intestinal Permeability and Susceptibility to Colitis. PLoS ONE 9(5): e98016. https://doi.org/10.1371/journal.pone.0098016
Editor: Christopher R. Weber, University of Chicago, United States of America
Received: December 7, 2013; Accepted: April 27, 2014; Published: May 20, 2014
Copyright: © 2014 Oh-oka et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by grants from the Ministry of Education, Culture, Sports, Science, and Technology, Japan (H25). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
The intestinal epithelium forms the boundary between the body and external environment and provides a selective permeable barrier that limits the permeation of luminal harmful molecules, while allowing the appropriate absorption of nutrients and water. This selective permeability in the intestine depends on specialized structures composed of tight junctions (TJs) –. TJs are multiple protein complexes consisting of Occludin, Zonula occludens (Zo), Claudins, and junctional adhesion molecules (JAM), located at the apical ends of the lateral membranes of epithelial cells , .
The circadian clock drives daily rhythms in physiology and behavior that enable organisms to keep track of the time of day –. Mammalian circadian clock consists of the central oscillator located in the suprachiasmatic nucleus (SCN) of the hypothalamus and peripheral oscillators present in nearly all cell types –. The central SCN clock is set by light and synchronizes the peripheral clocks via neural and endocrine pathways. At the cellular levels, the circadian clock is based on autonomous interlocking transcriptional-translational feedback loops consisting of several “clock genes”. Briefly, the PAS domain helix-loop-helix proteins Clock and Bmal1 bind as heterodimers to regulatory elements of Cryptochrome (Cry) and Period (Per) genes via E-box and stimulate the transcription of these genes. Once the repressor proteins Cry and Per have reached a critical concentration, they attenuate the activity of Clock-Bmal1 heterodimers and thereby repress the transcription of their own genes. These core molecular clocks also control expression of clock-controlled genes (CCGs) via direct or indirect transcriptional activation through clock-controlled transcriptional regulators.
Previous studies suggest that the circadian clock is present in intestinal epithelial cells and plays an important role in several functions of intestinal epithelial cells, such as retaining homeostasis, cell division and lipid absorption –. A recent study suggested that intestinal permeability was also regulated by the circadian clock . However, the precise mechanisms how the circadian clock affects intestinal permeability remain largely unknown. The primary aim of this study is to investigate whether the expression levels of TJ proteins that play an important role in intestinal permeability are under control of the circadian clock.
Female 5- to 6-week-old ICR mice purchased from Japan SLC (Tokyo, Japan), ICR mPer2m/m and ClockΔ19/Δ19 mice were bred under specific pathogen-free conditions. All mice were housed in 12-hours light/12-hours dark conditions (L/D cycles; the light was turned on at 6∶00 AM, Zeitgeber time [ZT] 0, and the light was turned off at 6∶00 PM, ZT12) with ad libitum access to food and water, for at least 2 weeks. All animal experiments were approved by the institutional review board of the University of Yamanashi and the Committee for Animal Experimentation of the School of Science and Engineering at Waseda University.
Quantitative Real-time PCR (Q-PCR)
A quantitative real-time PCR analysis using cDNA from the mouse tissue specimens was performed using the AB7300 real-time PCR system (Applied Biosystems, Foster City, CA, USA) according to the manufacturer’s instructions, using primers and probes for mouse Occludin, Zonula occludens (Zo)-1, Claudin-1, 2, 3, 4, 7, Junctional adhesion molecule (JAM), Myeloperoxidase (Mpo), Heme oxygenase (Ho)-1, Cyclooxygenase (Cox)-2, Prostaglandin E synthetase (Pges)-1, Per1, 2, 3, Clock, Bmal1, Cry1, IL-1β, TNF-α and Glyceraldehyde-3-phosphate dehydrogenase (GAPDH; Applied Biosystems) as previously described . The ratio of each gene to that of GAPDH was calculated, and the relative expression levels are shown.
Isolation of Intestinal Epithelial Cells (IECs)
IECs were isolated as described previously . Briefly, an inverted large intestine of mice was cut into four segments and the segments were transferred to a 50-ml tube containing 10 ml RPMI1640 medium/5% FCS/25mM Hepes. The tube was shaken at 37°C at 100 rpm for 45 min. After incubation, vortex the cell solution vigorously for 20s and pass through a 70 µm cell strainer. Subsequently, the cells were suspended in 30% (wt/vol) Percoll and centrifuged at 20°C at 400×g for 20 min. After centrifugation, cells at the top of the 30% Percoll were enriched with IECs.
Western Blot Analysis
IECs were lysed in buffer containing 0.1 M Tris-HCl (pH 7.5)/2% SDS/10% glycerol/5% 2-mercaptoethanol, boiled at 95°C for 5 min and centrifuged at 15000 rpm for 5 min and analyzed by reducing SDS-PAGE. Immunoblots were performed using antibodies against Claudin-1 (Abcam, Cambridge, UK, ab15098, 1∶100), Occludin (Applied Biosystems, 71–1500, 1∶250), Clock (Abcam, ab3517, 1∶200), Bmal1 (Abcam, ab3350, 1∶200) and β-actin (Santa Cruz, Texas, sc-1615, 1∶200). Quantitative analysis of Western blotting was done by using the Scion Image Software package and relative intensities of the target proteins to β-actin were shown.
Chromatin Immunoprecipitation (CHiP) Assay
CHiP was performed using SimpleChIP® Enzymatic Chromatin IP Kit (Agarose Beads) (Cell signaling, Tokyo, Japan, #9002). Isolated IECs or cultured cells (HCT116 and Caco-2) were cross-linked in 1% formaldehyde, followed by the CHiP assay according to the manufacturer’s protocol. Rabbit polyclonal antibodies to CLOCK (ab3517) and BMAL1 (ab3350) were from Abcam (Cambridge, UK). DNA was purified and Q-PCR was done using SYBRgreen reagent (QIAGEN, Hilden, Germany). Primers used were as follows: mouse Occludin promoter forward 5′-CTCCCATCCGAGTTTCAGGT-3′ and reverse 5′-GCTGTCGCCTAAGGAAAGAG-3′, mouse Claudin-1 promoter forward 5′-GTTTGCAGAGACCCCATCAC-3′ and reverse 5′-AGAAGCCAGGATGAAACCCA-3′, human Occludin promoter forward 5′-CTCCCATCCGAGTTTCAGGT-3′ and reverse, human Claudin-1 promoter forward 5′-CTCCCCGCCTTAACTTCCTC-3′ and reverse 5′-CAGGAAGGCGAGAATGAAGC-3′, 5′-GGAGTGTAGGTGTGGTGTGT-3′.
Isolation of Promoter Fragments and Reporter Assay
A human Occludin (–60 to +240) or Claudin-1 (–1010 to +360) promoter fragment containing E-box elements was amplified by PCR from genomic DNA of TIG120 cells and cloned into the pGL3 vector (Promega, Fitchburg, Wisconsin) upstream of firefly luciferase. Primer sequences used were as follows: human Occludin promoter forward 5′-CTCGAGAAAGTGCTGAGTGCCTGGAC-3′ and reverse 5′-AAGCTTTCGGTGACCAATTCACCTGA-3′, human Claudin-1 promoter forward 5′-CTCGAGGGAAACTACAGTCCCAGCGA-3′ and reverse 5′-AAGCTTGATGTTGTCGCCGGCATAGG-3′. These reporter constructs were transfected into HCT-116 or Caco-2 cells cultured in 96-well plates using Fugene6 (Promega) in the presence of pcDNA3, pcDNA3-Clock or pcDNA3-Bmal1 expression plasmid. pcDNA3-Clock and pcDNA3-Bmal1 expression plasmids were kindly gifted by Dr. Fuyuki Sato (Hirosaki University, Aomori, Japan). At 48 hours after transfection, the luciferase activity was measured using the luciferase reporter assay system (TOYO ink, Tokyo, Japan).
In vivo Colon Permeability Assay
Colon permeability to Evans blue was measured in vivo. This is a well-validated method for assessing epithelial permeability . In isoflurane-N2O anesthesia, spontaneously breathing animals were placed in a supine position on a heating pad and a laparotomy was performed. A small polyethylene tube (G22) was inserted into the proximal colon ascendens (immediately adjacent to the cecum) and secured by a ligature. Via this tube the colon was gently flushed until all stool was rinsed out, and 1 ml of Evans blue 1.5% (Sigma-Aldrich Japan, Tokyo, Japan, E2129) in PBS was instilled into the colon and left in place for 15 min. Then the colon was rinsed with PBS, until the perianal washout was clear. Animals were euthanized, and the colon was rapidly taken out. It was rinsed again with several milliliters of PBS, followed by 1 ml of 6 mM N-acetylcysteine to eliminate dye sticking in the colonic mucus. The colon was opened and rinsed once more with PBS, and its length was recorded. The whole colon was placed in 2 ml N,N-dimethylformamide for 12 h to extract the Evans blue dye. The dye concentration in the supernatant was measured spectrophotometrically at 610 nm and given as extinction per gram colonic tissue.
Ex vivo Colon Permeability Assay
Colon permeability was measured in isolated intestinal segments using FITC-dextran or Horseradish peroxidase (HRP) as described previously , . Briefly, 6-cm segments of the large intestine were removed, rinsed with ice-cold saline, everted, filled with 700 µl PBS and ligated at both ends. The filled colon segments were incubated in PBS containing 1 mM FITC-dextran (average mol wt, 4000 Sigma-Aldrich Co. Missouri, USA) or 0.4 mg/ml HRP (type IV, 300 units/mg solid, Sigma-Aldrich Co.). The colon sacs were removed after 45 minutes and the contents (∼500 µL) of each sac was collected carefully using a 1-ml syringe. The amount of FITC-dextran that transverse the colon was quantified by fluorescence plate reader at 521 nm. The HRP activity in the contents of each sac was determined spectrophotometrically based on the rate of oxidation of pyrogallol.
Induction of Dextran Sodium Sulfate (DSS)-induced Colitis
Colitis was induced by feeding mice 5% DSS dissolved in drinking distilled water (mol wt 5000; Wako Pure Chemical Co., Osaka, Japan) for 7 days, as previously described .
The stool scores were calculated based on a method by Cooper et al. with some modifications . Briefly, the scoring was based on a maximum score of 4. Stool consistency was graded as: 0 = firm, 1 = loose, 2 = diarrhea. Blood in the stool was also evaluated on a 0- to 2-point scale. 0 = no blood, 1 = occult blood, 2 = gross rectal bleeding.
Enzyme-Linked Immunosorbent Assay (ELISA)
The concentrations of tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) in the mouse colon tissue were determined using the mouse TNF-α and IL-6 enzyme-linked immunosorbent assay (ELISA) kits (R&D Inc, Minneapolis, MN), respectively, according to the manufacturer’s instructions.
Generation of Bone Marrow Chimeric Mice
Recipient wild-type mice or mPer2m/m mice were lethally irradiated with 1050 rads using a Co irradiator and then were injected intravenously 6 hours later with 1×107 bone marrow cells derived from the femurs of the respective donors.
Values represent the mean ± S.D. The statistical analysis was performed using the unpaired Studentns t-test. The statistical analysis of time-of-day-dependent variations in mRNA and protein levels was performed using a one-way analysis of variance (ANOVA). A value of P<0.05 was considered to be significant.
Occludin and Claudin-1 Expressions Exhibit a Time of Day-dependent Variation in Colonic Epithelium, Relying on Normal Per2 Activity
The mRNA levels of Occludin, Zo-1, Claudin-1, -2, -3, -4, -7 and JAM-A, major components of TJ, were examined in the colon tissue samples obtained from wild-type mice and mice with the loss-of-function mutation of a key clock gene Period2 (Per2; mPer2m/m) every 6 hours during the day to investigate the possible circadian variations . We found that the mRNA levels of Occludin and Claudin-1, but not Claudin-2, -3, -4, -7, Zo-1, and JAM-A, in the colon showed a time of day-dependent variation in wild-type mice (Fig. 1A). Detailed kinetic analysis of mRNA levels of Occludin and Claudin-1 at every 4 hours during the day revealed that the mRNA levels showed a nadir at Zeitgeber time (ZT) 12 (18∶00 PM) (Fig. S1A). We also found that Per1, Per2, Cry1, and Bmal1 mRNA levels in the colon exhibited a time of day-dependent variation (Fig. S2) and the kinetics of Per1 and Per2 mRNA expression were the opposite phase to Occludin and Claudin-1 mRNA kinetics (Fig. 1A). Consistently, the expression levels of Occludin and Claudin-1 proteins in colonic IECs showed a similar time of day-dependent variation to the mRNA expressions (Fig. 1B and C). Detailed kinetic analysis of protein levels of Occludin and Claudin-1 at every 4 hours during the day revealed that the protein levels showed a nadir at ZT 16 (22∶00 PM) (Fig. S1B), suggesting that there was a time-lag between mRNA and protein expression changes of Occludin and Claudin-1. However, please note that the circadian changes of Occludin, but not Claudin-1, protein levels did not reach a statistical significance (p = 0.053) and showed only a trend of circadian oscillations in this analysis. In contrast to wild-type mice, mPer2m/m mice showed constitutively high expression levels of Occludin and Claudin-1 mRNAs and proteins without oscillations (Fig. 1). These results suggested that Occludin and Claudin-1 expressions in colonic epithelium showed circadian oscillations, which relied on normal Per2 activity.
(A) Real-time PCR analysis for Occludin, Zo-1, Claudin-1, -2, -3, -4, -7, and JAM mRNAs expression in the colon tissue samples obtained from the wild-type mice and mPer2m/m mice at the indicated time points (n = 8 per group). (B) Representative pictures of Western blot for Occludin, Claudin-1 and β-actin expression in colonic epithelial cell samples from wild-type and mPer2m/m mice obtained at the indicated time points (n = 3 per group). (C) The quantitative analysis of (B) (n = 3 per group). *p<0.05.
Clock and Bmal1 Bind to the E-box Element in the Promoter Regions of Occludin and Claudin-1 Genes and Affect their Trancriptional Responses
The Clock-Bmal1 heterodimer binds directly to the E-box element of the clock controlled genes (CCGs) , . Occludin and Claudin-1 possess E-box elements in the promoter regions of the genes , . To examine whether Clock and Bmal1 bind to the E-box elements of Occludin and Claudin-1 promoter regions, we performed CHiP assays by using IECs isolated from the colonic tissues of wild-type and mPer2m/m mice every 12 hours during the day.
Clock and Bmal1 bound to the E-box elements of Occludin and Claudin-1 promoter regions in mouse colonic IECs (Fig. 2A). Interestingly, the ratio of binding showed a time of day-dependent variation in IECs from the colon of wild-type mice, with a high at day (ZT4: 10∶00 AM) and low at night (ZT16: 10∶00 PM) (Fig. 2A). However, such a variation was absent in mPer2m/m mice (Fig. 2A). The bindings of Clock and Bmal1 to the E-box elements of Occludin and Claudin-1 promoter regions were also confirmed in human colonic epithelial cell lines HCT-116 and Caco-2 cells (Fig. 2B and Fig. S3A).
(A) CHiP assays for recruitments of Clock and Bmal1 to E-box elements in Occludin and Claudin-1 promoter region using colonic IECs obtained at the indicated time points from wild-type mice and mPer2m/m mice (n = 3 per group). Similar results were obtained in two independent experiments. (B) CHiP assays for recruitments of CLOCK and BMAL1 to E-box elements in Occludin and Claudin-1 promoter region using HCT116 cells (n = 3 per group). (C) Relative luciferase activity in HCT116 cells transfected with Occludin and Claudin-1 promoter reporter plasmids together with Clock and Bmal1 expression vectors or a control empty vector (n = 3 per group). *p<0.05.
To investigate whether Clock and Bmal1 regulate transcriptional responses of Occludin and Claudin-1, we performed reporter assays using luciferase-reporter plasmids containing human Occludin (–60 to +240) or Claudin-1 (–1010 to +360) promoter fragments with E-box elements. Transfection of the Occludin- or Claudin-1 promoter reporter plasmids into HCT-116 cells showed ∼20-fold increases in luciferase activity compared with transfection of control pGL3-basic plasmid without containing the promoter elements (data not shown), suggesting that these plasmids were activated in HCT-116 cells. Overexpression of Clock or Bmal1 in HCT-116 cells further increased the luciferase activity of Occludin- or Claudin-1-reporter plasmids (Fig. 2C). Similar findings were also observed in Caco-2 cells (Fig. S3B). These results suggested that Clock and Bmal1 bound directly to the E-box elements of Occludin and Claudin-1 promoters and increased the transcriptional responses.
Colonic Permeability is under the Circadian Control, which is Inversely Associated with Occludin and Claudin-1 Expression Levels
The possibility of altered epithelial permeability in wild-type and mPer2m/m mice was investigated since the TJ proteins play an important role in epithelial permeability through the control of the paracellular pathway for movement of intestinal fluid and macromolecules –. Colonic permeability to Evans blue in vivo and to FITC-dextran and HRP ex vivo showed a time-of-day dependent variation in wild-type mice, with a high at night (ZT16: 10∶00 PM) and low at day (ZT4: 10∶00 AM), which was the opposite phase to Occludin and Claudin-1 protein levels (Fig. 2 and 3). However, such a variation was absent in mPer2m/m mice and, as expected, the colonic permeability was lower in mPer2m/m mice than that in wild-type mice (Fig. 3). These results suggested that colonic permeability was under control of the circadian clock, which was associated with temporal alterations of colonic permeability.
(A) Permeability to evans blue in vivo at the indicated time points from wild-type mice and mPer2m/m mice (n = 5 per group). (B) Permeability to FITC-dextran 4000 ex vivo obtained at the indicated time points from wild-type mice and mPer2m/m mice (n = 6 per group). (C) Permeability to HRP ex vivo obtained at the indicated time points from wild-type mice and mPer2m/m mice (n = 8 per group). *p<0.05.
mPer2m/m Mice Show Reduced Susceptibility to DSS-induced Colitis
We then investigated whether mPer2m/m mice with reduced colonic permeability were more resistant to colonic injury induced by DSS, because intestinal permeability was associated with susceptibility to colitis –, .
mPer2m/m mice given 5% DSS in drinking water over a period of 7 days showed less body weight loss, bloody stool, and colon shrinkage than the wild-type mice. Reduced levels of TNF-α and IL-6 proteins in the colon were observed in the mPer2m/m mice in comparison to wild-type mice (Fig. 4A–D). The mRNA expression levels of pro-inflammatory molecular markers, myeloperoxidase (Mpo), heme oxygenase (Ho)-1, cyclooxygenase (Cox)-2, and prostaglandin E synthase (Pges)-1 in the colon were also less in mPer2m/m mice than those in wild-type mice (Fig. S4A). mPer2m/m mice drank equivalent amounts of DSS-containing water to wild-type mice per day during the period of DSS challenge (day 0–7; Fig. S4B). These results suggested that mPer2m/m mice were more resistant to DSS-induced colitis in comparison to wild-type mice.
(A) Body weight changes in mice for 6 days after administration of 5% DSS. (B) Stool scores in mice for 6 days after administration of 5% DSS. (C) Colon length in mice on day 7 after administration of 5% DSS. (D) TNF-α and IL-6 levels in the colon tissue samples measured by ELISA. The samples were obtained from the mice on day 7 after administration of 5% DSS. The susceptibility to DSS-colitis in wild/mPer2m/m bone marrow chimeras mice was shown (E–H). (E) Body weight changes in mice 6 days after administration of 5% DSS. (F) Stool scores in mice 6 days after administration of 5% DSS. (G) Colon length in mice on day 7 after administration of 5% DSS. (H) IL-6 levels measured by ELISA in the colon tissue samples obtained from the mice on day 7 after the administration of 5% DSS. n = 8 per group, *p<0.05. The mice were sacrificed at ZT4 (10∶00 AM) in Fig. 5C, D, G and H.
Bone marrow chimeric mice in which the donors and/or the recipients were either wild-type or mPer2m/m mice were generated to further investigate whether the Per2 mutation in the tissue resident cells (most likely colonic epithelium) was important for the protection against DSS-induced colitis. DSS challenge of chimeric mice lacking the Per2 mutation in hematopoietic cells induced pro-inflammatory markers similar to those observed in wild-type mice reconstituted with wild-type bone marrow cells (Fig. 4E–H, Fig. S5). In contrast, chimeric mice lacking Per2 in the non-hematopoietic cells were resistant to DSS-induced colitis similar to the mPer2m/m mice reconstituted with mPer2m/m bone marrow cells, as indicated by the changes of those parameters (Fig. 4E–H, Fig. S5). These results suggested that the Per2 mutation conferred protection against DSS-induced colitis when it occurred in non-hematopoietic cells (presumably the cells of the colonic epithelium).
ClockΔ19/Δ19 Mice Show Enhanced Susceptibility to DSS-induced Colitis
To further ascertain that the circadian clock regulated a time of day-dependent variation of Occludin and Claudin-1expression in the colon, we examined the mRNA levels of Occludin and Claudin-1 in the colon tissue samples obtained from wild-type mice and mice with the loss-of-function mutation of another key clock gene Clock (ClockΔ19/Δ19 mice) every 6 hours during the day. The mRNA levels of Occludin and Claudin-1 in the colon showed a time of day-dependent variation in wild-type mice whereas the variation was absent in ClockΔ19/Δ19 mice (Fig. S6A). Interestingly, the mRNA levels of Occludin and Claudin-1 in the colon were relative low in ClockΔ19/Δ19 mice compared with those in wild-type mice. Consistently, ClockΔ19/Δ19 mice were more sensitive to the colonic injury induced by DSS than wild-type mice as judged by body weight changes, stool property, and Ho-1 and Pges-1 expression (Fig. S6B–D). These results also suggested that disruption of the circadian clock affected TJ protein expression and barrier function. The opposite phenotypes observed in ClockΔ19/Δ19 mice to those in mPer2m/m mice may be attributed to the opposite function of Clock and Per2 in the circadian clockwork; Clock acts as a positive limb whereas Per2 acts as a negative limb in the transcriptional-translational feedback loop in the molecular clock machinery.
IL-1β and TNF-α mRNAs Expression does not Exhibit a Time of Day-dependent Variation in the Mouse Colon
Major proinflammatory cytokines IL-1β and TNF-α have been shown to regulate epithelial barrier function . To investigate whether the circadian clock also affects IL-1β and TNF-α expression, we examined mRNA levels of IL-1β and TNF-α in the colon tissue samples obtained from wild-type mice and mPer2m/m mice every 6 hours during the day. We found that IL-1β and TNF-α mRNA levels did not show circadian oscillations in wild-type mice and they were comparable between wild-type and mPer2m/m mice (Fig. S7).
The circadian clock plays an important role in the regulation of several physiological functions of the intestine as well as other organs –. Most recently, Summa et al. also suggested that the circadian clock regulated intestinal permeability although the precise mechanisms remain largely unknown . The current study demonstrated that the expressions of TJ proteins Occludin and Claudin-1 in the large intestinal epithelium showed daily oscillations, which required normal Per2 activity and the temporal changes of the expression levels were inversely associated with colonic permeability and different susceptibility to DSS-induced colitis between wild-type and mPer2m/m mice. These results suggest that the circadian clock regulates a time of day-dependent variation in colonic TJ protein expression levels, thereby affecting intestinal permeability on a daily basis and also susceptibility to colitis.
One of the primary findings of this study was that 1) Occludin and Claudin-1 mRNAs showed daily oscillations, 2) Clock and Bmal1 bond directly to the E-box elements of Occludin and Claudin-1 promoters, and 3) Overexpression of Clock and Bmal1 activated Occludin and Claudin-1 promoters containing E-box elements (Fig. 1 and 2). These results suggest that Occludin and Claudin-1 are CCGs with E-box elements in the promoter region directly regulated by Clock/Bmal1 heterodimers. However, a recent study suggests that only 22% of mRNA cycling genes are driven by de novo transcription in mouse liver . Thus, we can not exclude the possibility that posttranscriptional mechanisms may also underlie the circadian regulation of Occludin and Claudin-1 expression.
We showed that colonic permeability was under the circadian control, which was inversely associated with Occludin and Claudin-1 expression levels (Fig. 3). Claudins are defined as most important key factors for tight junction permeability and the tightness of TJ is determined by the number, composition, and the mixing ratio of Claudins –. Recent studies also suggest that Occludin is important for the maintenance and assembly of TJ proteins –.
We also found that IL-1β and TNF-α mRNA levels did not show circadian oscillations in wild-type mice and they were comparable between wild-type and mPer2m/m mice (Fig. S7). Therefore, it is likely that Clock-Bmal1 heterodimers regulate colonic permeability in a circadian manner by timing the expression level of Claudin-1 and Occludin and affect TJ function. However, it remains to be determined whether Occludin and Claudin-1 expression change are indeed primarily responsible for daily changes of tight junction permeability and susceptibility to DSS-induced colitis.
It appears that the current findings are incompatible with the previous findings by Summa et al. . They reported that there were no significant differences in Occludin mRNA expression in the colon between wild-type mice and mice with a mutation of Clock (ClockΔ19/Δ19 mice) and there were increased cytoplasmic (i.e. decreased membrane-bound) Occludin protein levels in ClockΔ19/Δ19 mice compared with those in wild-type mice . However, they examined Occludin mRNA and cytoplasmic Occludin protein levels in the mouse colon at only one time point in wild-type and ClockΔ19/Δ19 mice. They did not examine total Occludin protein levels, either. These experimental differences might explain the discrepancy between the current and previous findings. Actually, we found that the mRNA levels of Occludin and Claudin-1 in the colon showed a time of day-dependent variation in wild-type mice whereas the variation was absent in ClockΔ19/Δ19 mice (S-Fig. 6A).
In addition, Summa et al. reported that ClockΔ19/Δ19 mice had increased colonic permeability compared with wild-type mice . Since Clock (a positive limb of the circadian clock machinery) and Per2 (a negative limb of the circadian clock machinery) functions as an opposite direction as “clock genes”, it may be that ClockΔ19/Δ19 mice have opposite circadian phenotypes to mPer2m/m mice. The current findings that ClockΔ19/Δ19 mice were sensitive to DSS-induced colonic injury compared to wild-type mice in contrast to mPer2m/m mice (Fig. S6) may support this idea.
A dysfunction of TJ proteins weakens intercellular adhesion, promotes intestinal permeability, and inflammation , . Therefore, constitutively high Occludin and Claudin-1 protein expression levels in the large intestine are likely to be attributed the reduced susceptibility to DSS-induced colitis in mPer2m/m mice (Fig. 4). Although, it is possible that the reduced susceptibility to DSS-induced colitis in mPer2m/m mice may be due to some type of immune cell dysfunction, such as NK cells and macrophages, the chimeric mouse experiments strongly suggest that the Per2 mutation in non-hematopoietic cells is responsible for the protection against DSS-induced colitis (Fig. 4).
It is not clear about the half-life of Occludin and Claudin-1 proteins in vivo. In vitro studies showed that basal half-life of Occludin was about ∼6 hours in primary human brain microvascular cells  and basal half-life of Claudin-1 was also about ∼6 hours in human lung carcinoma cell line A549 cells . Hence, we speculate that Occludin and Claudin-1 proteins might have relatively short half-life in the mouse colon and circadian regulation of these molecules at the transcriptional levels could indeed contribute to the changes of the protein levels in vivo.
In summary, we suggest that expressions of TJ proteins Occludin and Claudin-1 in the large intestinal epithelium are under control of the circadian clock, which is associated with temporal changes of colonic permeability and also susceptibility to colitis. To our knowledge, this is the first study showing a novel regulation of TJs by the circadian clock. The findings would provide new insights into the regulation of intestinal barrier function and pathophysiology of colitis.
Occludin and Claudin-1 expressions exhibit a time of day-dependent variation in colonic epithelium (detailed kinetic analysis). (A) Real-time PCR analysis for Occludin and Claudin-1 mRNAs expression in the colonic epithelial cells samples obtained at the indicated time points from wild-type mice (n = 4 per group). (B) The quantitative analysis of Western blot pictures for Occludin and Claudin-1 expression in the colonic epithelial cell samples from wild-type mice obtained at the indicated time points (n = 4 per group).
Kinetics of major “clock genes” mRNAs expression in the mouse colon. Real-time PCR analysis for Per1, Per2, Per3, Clock, Bmal1 and Cry1 mRNAs expression in the colon tissue samples from wild-type mice and mPer2m/m mice obtained at the indicated time points (n = 8 per group).
Clock and Bmal1 bind to the E-box element in the promoter regions of Occludin and Claudin-1 genes and affect their transcriptional responses in Caco-2. (A) CHiP assays for recruitments of Clock and Bmal1 to E-box elements in Occludin and Claudin-1 promoter region using Caco-2 cells (n = 3 per group). (B) Relative luciferase activity in Caco-2 cells transfected with Occludin and Claudin-1 promoter reporter plasmids together with Clock and Bmal1 expression vectors or a control empty vector (n = 3 per group). *p<0.05.
mPer2m/m mice show reduced susceptibility to DSS-induced colitis. (A) Real-time PCR analysis for Mpo, Ho-1, Cox-2 and Pges1 mRNAs expression in the colon tissue obtained from the mice on day 7 after the administration of 5% DSS. (B) The amounts of drinking water in mice for 6 days after administration of 5% DSS. n = 8 per group, *p<0.05. The mice were sacrificed at ZT4 (10∶00 AM).
Per2 mutation in non-hematopoietic cells is critical for protection against DSS-induced colitis. Real-time PCR analysis for Ho-1, Cox-2 and Pges-1 mRNAs expression in the colon tissue obtained from the mice on day 7 after administration of 5% DSS. n = 8 per group, *p<0.05. The mice were sacrificed at ZT4 (10∶00 AM).
ClockΔ19/Δ19 mice show enhanced susceptibility to DSS-induced colitis. (A) Real-time PCR analysis for Occludin and Claudin-1 mRNAs expression in the colon tissue samples from wild-type mice and ClockΔ19/Δ19 mice obtained at the indicated time points (n = 4 per group). (B–D) The severity of DSS-colitis in the wild-type and ClockΔ19/Δ19 mice; Body weight changes in mice for 5 days after administration of 5% DSS (B). Stool scores in mice for 5 days after administration of 5% DSS (C). Real-time PCR analysis for Ho-1 and Pges1 mRNAs expression in the colon tissue obtained from the mice on day 6 after the administration of 5% DSS (D). n = 4 per group, *p<0.05. The mice were sacrificed at ZT4 (10∶00 AM).
Kinetics of IL-1β and TNF-α mRNAs expression in the mouse colon. Real-time PCR analysis for IL-1β and TNF-α mRNAs expression in the colon tissue samples from wild-type mice and mPer2m/m mice obtained at the indicated time points (n = 4 per group).
We thank Ms. Natsumi Aoki, Tomoko Tohno, Yuko Ohnuma, and Mutsuko Hara for their assistance, and Dr. Fuyuki Sato (Hirosaki University, Aomori, Japan) for providing us with human Clock and Bmal1 expression plasmids.
Conceived and designed the experiments: K. Oh-oka AN. Performed the experiments: K. Oh-oka HK KI. Analyzed the data: K. Oh-oka HK KI. Contributed reagents/materials/analysis tools: HO K. Okumura SS AN. Wrote the paper: K. Oh-oka AN.
- 1. Groschwitz KR, Hogan SP (2009) Intestinal barrier function: molecular regulation and disease pathogenesis. J Allergy Clin Immunol 124: 3–20.
- 2. Suzuki T (2013) Regulation of intestinal epithelial permeability by tight junctions. Cell Mol Life Sci 70(4): 631–659.
- 3. Turner JR (2009) Intestinal mucosal barrier function in health and disease. Nat Rev Immunol 9: 799–809.
- 4. Dibner C, Schibler U, Albrecht U (2010) The mammalian circadian timing system: organization and coordination of central and peripheral clocks. Annu Rev Physiol 72: 517–549.
- 5. Takahashi JS, Hong HK, Ko CH, McDearmon EL (2008) The genetics of mammalian circadian order and disorder: implications for physiology and disease. Nat Rev Genet 9: 764–775.
- 6. Hogenesch JB, Ueda HR (2011) Understanding systems-level properties: timely stories from the study of clocks. Nat Rev Genet 12: 407–416.
- 7. Mukherji A, Kobiita A, Ye T, Chambon P (2013) Homeostasis in intestinal epithelium is orchestrated by the circadian clock and microbiota cues transduced by TLRs. Cell 153(4): 812–827.
- 8. Hoogerwerf WA (2006) Biologic clocks and the gut. Curr Gastroenterol Rep 8(5): 353–359.
- 9. Pan X, Munshi MK, Iqbal J, Queiroz J, Sirwi AA, et al. (2013) Circadian regulation of intestinal lipid absorption by apolipoprotein AIV involves forkhead transcription factors A2 and O1 and microsomal triglyceride transfer protein. J Biol Chem 288(28): 20464–20476.
- 10. Summa KC, Voigt RM, Forsyth CB, Shaikh M, Cavanaugh K, et al. (2013) Disruption of the Circadian Clock in Mice Increases Intestinal Permeability and Promotes Alcohol-Induced Hepatic Pathology and Inflammation. PLoS One 8(6): e67102.
- 11. Takamura T, Harama D, Matsuoka S, Shimokawa N, Nakamura Y, et al. (2010) Activation of the aryl hydrocarbon receptor pathway may ameliorate dextran sodium sulfate-induced colitis in mice. Immunol Cell Biol 88: 685–689.
- 12. Komano H, Fujiura Y, Kawaguchi M, Matsumoto S, Hashimoto Y, et al. (1995) Homeostatic regulation of intestinal epithelia by intraepithelial gamma delta T cells. Proc Natl Acad Sci U S A 92(13): 6147–6151.
- 13. Mennigen R, Nolte K, Rijcken E, Utech M, Loeffler B, et al. (2009) Probiotic mixture VSL#3 protects the epithelial barrier by maintaining tight junction protein expression and preventing apoptosis in a murine model of colitis. Am J Physiol Gastrointest Liver Physiol 296(5): G1140–1149.
- 14. Kono H, Fujii H, Asakawa M, Yamamoto M, Matsuda M, et al. (2003) Protective effects of medium-chain triglycerides on the liver and gut in rats administered endotoxin. Ann Surg 237: 246–255.
- 15. Bishop BL, Lodolce JP, Kolodziej LE, Boone DL, Tang WJ (2010) The role of anthrolysin O in gut epithelial barrier disruption during Bacillus anthracis infection. Biochem Biophys Res Commun 394(2): 254–259.
- 16. Okayasu I, Hatakeyama S, Yamada M, Ohkusa T, Inagaki Y, et al. (1990) A novel method in the induction of reliable experimental acute and chronic ulcerative colitis in mice. Gastroenterology 98: 694–702.
- 17. Cooper HS, Murthy SN, Shah RS, Sedergran DJ (1993) Clinicopathologic study of dextran sulfate sodium experimental murine colitis. Lab Invest 69: 238–249.
- 18. Zheng B, Larkin DW, Albrecht U, Sun ZS, Sage M, et al. (1999) The mPer2 gene encodes a functional component of the mammalian circadian clock. Nature 400: 169–173.
- 19. Oishi K, Miyazaki K, Kadota K, Kikuno R, Nagase T, et al. (2003) Genome-wide expression analysis of mouse liver reveals CLOCK-regulated circadian output genes. J Biol Chem 278(42): 41519–41527.
- 20. Storch KF, Lipan O, Leykin I, Viswanathan N, Davis FC, et al. (2002) Extensive and divergent circadian gene expression in liver and heart. Nature 417(6884): 78–83.
- 21. Ikenouchi J, Matsuda M, Furuse M, Tsukita S (2003) Regulation of tight junctions during the epithelium-mesenchyme transition: direct repression of the gene expression of claudins/occludin by Snail. J Cell Sci 116(Pt 10): 1959–1967.
- 22. Martínez-Estrada OM, Cullerés A, Soriano FX, Peinado H, Bolós V, et al. (2006) The transcription factors Slug and Snail act as repressors of Claudin-1 expression in epithelial cells. Biochem J 394(Pt 2): 449–457.
- 23. Henderson P, van Limbergen JE, Schwarze J, Wilson DC (2011) Function of the intestinal epithelium and its dysregulation in inflammatory bowel disease. Inflamm Bowel Dis 17: 382–395.
- 24. Koike N, Yoo SH, Huang HC, Kumar V, Lee C, et al. (2012) Transcriptional architecture and chromatin landscape of the core circadian clock in mammals. Science 338(6105): 349–354.
- 25. Furuse M, Fujita K, Hiiragi T, Fujimoto K, Tsukita S (1998) Claudin-1 and -2: novel integral membrane proteins localizing at tight junctions with no sequence similarity to occludin. J Cell Biol 141(7): 1539–1550.
- 26. Furuse M, Sasaki H, Tsukita S (1999) Manner of interaction of heterogeneous claudin species within and between tight junction strands. J Cell Biol 147(4): 891–903.
- 27. Nitta T, Hata M, Gotoh S, Seo Y, Sasaki H, et al. (2003) Size-selective loosening of the blood-brain barrier in claudin-5-deficient mice. J Cell Biol 161(3): 653–660.
- 28. Saitou M, Fujimoto K, Doi Y, Itoh M, Fujimoto T, et al. (1998) Occludin-deficient embryonic stem cells can differentiate into polarized epithelial cells bearing tight junctions. J Cell Biol 141(2): 397–408.
- 29. Furuse M, Itoh M, Hirase T, Nagafuchi A, Yonemura S, et al. (1994) Direct association of occludin with ZO-1 and its possible involvement in the localization of occludin at tight junctions. J Cell Biol 127(6 Pt 1): 1617–1626.
- 30. Saitou M, Furuse M, Sasaki H, Schulzke JD, Fromm M, et al. (2000) Complex phenotype of mice lacking occludin, a component of tight junction strands. Mol Biol Cell 11(12): 4131–4142.
- 31. Ramirez SH, Fan S, Dykstra H, Rom S, Mercer A, et al. (2013) Inhibition of glycogen synthase kinase 3β promotes tight junction in brain endothelial cells by half-life extension of Occudin and Clausin-5. PLoS ONE 8: e55972.
- 32. Kovalenko OV, Yang XH, Hemler ME (2007) A novel cysteine cross-linking method reveals a direct association between Claudin-1 and tetaraspanin CD9. Mol Cell Proteomics 6: 1855–1867.