Restricted feeding induces daily expression of clock genes and Pai-1 mRNA in the heart of Clock mutant mice

Plasminogen activator inhibitor-1 (PAI-1) is a key factor of fibrinolytic activity. The activity and mRNA abundance show a daily rhythm. To elucidate the mechanism of daily Pai-1 gene expression, the expression of Pai-1 and several clock genes was examined in the heart of homozygous Clock mutant (Clock/Clock) mice. Damping of the daily oscillation of Pai-1 gene expression in Clock/Clock mice was accompanied with damped or attenuated oscillations of mPer1, mPer2, mBmal1, and mNpas2 mRNA. Daily restricted feeding induced a daily mRNA rhythm of all clock genes and Pai-1 mRNA in Clock/Clock mice as well as wild-type mice. The peaks of clock genes and Pai-1 mRNA were phase-advanced in the heart of both genotypes after 6 days of restricted feeding. The present results demonstrate that daily Pai-1 gene expression depends on clock gene expression in the heart and that a functional Clock gene is not required for restricted feeding-induced resetting of the peripheral clock.     

Differences in the translation efficiency and mRNA stability mediated by 5'-UTR splice variants of human SP-A1 and SP-A2 genes

Surfactant protein A (SP-A) plays an important role in host defense, modulation of inflammatory processes, and surfactant-related functions of the lung. The human SP-A (hSP-A) locus consists of two functional genes, SP-A1 and SP-A2. Several hSP-A 5'-untranslated region (UTR) splice variants for each gene have been characterized and shown to be translated in vitro and in vivo. In this report, we investigated the role of hSP-A 5'-UTR splice variants on SP-A production and molecular mechanisms involved. We used in vitro transient expression of hSP-A 5'-UTR constructs containing luciferase as the reporter gene and quantitative real-time PCR to study hSP-A 5'-UTR-mediated gene expression. We found that 1) the four (A'D', ABD, AB'D', and A'CD') 5'-UTR splice variants under study enhanced gene expression, by increasing luciferase activity from 2.5- to 19.5-fold and luciferase mRNA from 4.3- to 8.8-fold compared with the control vector that lacked hSP-A 5'-UTR; 2) all four 5'-UTR splice variants studied regulated mRNA stability. The ABD variant exhibited the lowest rate of mRNA decay compared with the other three constructs (A'D', AB'D', and A'CD'). These three constructs also exhibited significantly lower rate of mRNA decay compared with the control vector; 3) based on the indexes of translational efficiency (luciferase activity/mRNA), ABD and AB'D' exhibited higher translational efficiency compared with the control vector, whereas the translational efficiency of each A'D' and A'CD' was lower than that of the control vector. These findings indicate that the hSP-A 5'-UTR splice variants play an important role in both SP-A translation and mRNA stability.     

Presence of robust circadian clock oscillation under constitutive over-expression of mCry1 in rat-1 fibroblasts

In mammals, mCRY proteins are essential and are major negative elements in circadian feedback loops. In this study, robust circadian clock oscillation was present even under conditions with constitutive over-expression of mCry1 in rat-1 cells. Rat-1 cells were produced to stably express mPer2 promoter-driven luciferase reporter, in which mCry1 was overexpressed under a tetracycline-dependent gene expression (Tet-On) system. Using these cells, we show that circadian clock oscillations in rat-1 fibroblasts persist when the mCRY1 protein constitutively accumulates in the nuclei.     

Analysis of a Charcot-Marie-Tooth disease mutation reveals an essential internal ribosome entry site element in the connexin-32 gene

A mutation located in the 5'-untranslated region (5'-UTR) of the nerve-specific connexin-32 mRNA, previously found in a family with Charcot-Marie-Tooth disease (CMTX), was analyzed for its effect on the expression of a reporter gene (luciferase) in transgenic mice and in transfected cells. Whereas both mutant and wild-type genes appeared to be transcribed and spliced efficiently, no luciferase was detected from the mutant in either system, suggesting that the mutation affects translation of the mRNA. When the 5'-UTR of nerve-specific connexin-32 mRNA was inserted between the two genes of a bicistronic vector and transfected into various cell lines, expression of the second gene was significantly increased. Because the mutant did not facilitate translation of the second gene in the bicistronic mRNA system, this result suggested that the CMTX mutation abolished function of an internal ribosome entry site (IRES) in the 5'-UTR of the wild-type connexin-32 mRNA. The CMTX phenotype of the mutant 5'-UTR further suggested that the wild-type IRES was essential for the translation of the connexin-32 mRNA in nerve cells. In addition, other sequence elements of the connexin-32 IRES were characterized by mutation analysis. A mutation in either of the first two elements investigated showed loss of IRES function, whereas mutation of a third element showed gain of function.     

The molecular mechanism regulating the autonomous circadian expression of Topoisomerase I in NIH3T3 cells

To identify whether Topoisomerase I (TopoI) has autonomous circadian rhythms regulated by clock genes, we tested mouse TopoI (mTopoI) promoter oscillation in NIH3T3 cells using a real-time monitoring assay and TopoI mRNA oscillations using real-time RT-PCR. Analysis of the mTopoI promoter region with Matlnspector software revealed two putative E-box (E1 and E2) and one DBP/E4BP4-binding element (D-box). Luciferase assays indicated that mTopoI gene expression was directly regulated by clock genes. The real-time monitoring assay showed that E-box and D-box response elements participate in the regulation of the circadian expression of mTopoI. Furthermore, a gel-shift assay showed that E2 is a direct target of the BMAL1/CLOCK heterodimer and DBP binds to the putative D-site. These results indicate that TopoI is expressed in an autonomous circadian rhythm in NIH3T3 cells.     

Serine 714 might be implicated in the regulation of the phosphorylation in other areas of mPer1 protein

The phosphorylation of mPer proteins may play important roles in the mechanism of the circadian clock via changes in subcellular localization and degradation. However, the mechanism has remained unclear. Previously, we identified three putative casein kinase (CK)1epsilon phosphorylation motif clusters in mPer1. In this work, we examined the role of the phosphorylation of serine residue, Ser(S)714, in mPer1. mPer1 S[714-726]A mutant, in which potential phosphorylation serine residues replaced by alanine residues, is rapidly phosphorylated compared with wild-type mPer1 by CK1epsilon. Coexpression with S[714]G mutant of mPer1 advanced phase of circadian expression of mPer2-luc expression, which was monitored by in vitro bioluminescence system. This result showed that the mPER1 S[714]G mutation affects circadian core oscillator. Considering these, it seems that Ser 714 might be involved in the regulation of the phosphorylation of other sites in mPer1 by CK1epsilon.     

MPer1 and mper2 are essential for normal resetting of the circadian clock

Mammalian Per1 and Per2 genes are involved in the mechanism of the circadian clock and are inducible by light. A light pulse can evoke a change in the onset of wheel-running activity in mice by shifting the onset of activity to earlier times (phase advance) or later times (phase delays) thereby advancing or delaying the clock (clock resetting). To assess the role of mouse Per (mPer) genes in circadian clock resetting, mice carrying mutant mPer1 or mPer2 genes were tested for responses to a light pulse at ZT 14 and ZT 22, respectively. The authors found that mPer1 mutants did not advance and mPer2 mutants did not delay the clock. They conclude that the mammalian Per genes are not only light-responsive components of the circadian oscillator but also are involved in resetting of the circadian clock.     

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

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.

Methods

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; mPer2^m/m). In addition, the susceptibility to dextran sodium sulfate (DSS)-induced colitis was compared between wild-type mice and mPer2^m/m mice.

Results

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 mPer2^m/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 mPer2^m/m mice. mPer2^m/m mice were more resistant to the colonic injury induced by DSS than wild-type mice.

Conclusions

Occludin and Claudin-1 expressions in the large intestine are under the circadian control, which is associated with temporal regulation of colonic permeability and also susceptibility to colitis.