Nmu/Nms/Gpr176 Triple-Deficient Mice Show Enhanced Light-Resetting of Circadian Locomotor Activity.

The suprachiasmatic nucleus (SCN) is the master circadian clock in mammals and is properly entrained by environmental light cycle. However, the molecular mechanism(s) determining the magnitude of phase shift by light is still not fully understood. The orphan G-protein-coupled receptor Gpr176 is enriched in the SCN, controls the pace (period) of the circadian rhythm in behavior but is not apparently involved in the light entrainment; Gpr176-/- animals display a shortened circadian period in constant darkness but their phase-resetting responses to light are normal. Here, we performed microarray analysis and identified enhanced mRNA expression of neuromedin U (Nmu) and neuromedin S (Nms) in the SCN of Gpr176-/- mice. By generating C57BL/6J-backcrossed Nmu/Nms/Gpr176 triple knockout mice, we noted that the mutant mice had a greater magnitude of phase shift in response to early subjective night light than wildtype mice, while Nmu/Nms double knockout mice as well as Gpr176 knockout mice are normal in the phase shifts induced by light. At the molecular level, Nmu-/-Nms-/-Gpr176-/- mice had a reduced induction of Per1 and cFos mRNA expression in the SCN by light and mildly upregulated circadian expression of Per2, Prok2, Rgs16, and Rasl11b. These expressional changes may underlie the phenotype of the Nmu/Nms/Gpr176 knockout mice. Our data argue that there is a mechanism requiring Nmu, Nms, and Gpr176 for the proper modulation of light-induced phase shift in mice. Simultaneous modulation of Nmu/Nms/Gpr176 may provide a potential target option for modulating the circadian clock.

Circadian protection against bacterial skin infection by epidermal CXCL14-mediated innate immunity.

The epidermis is the outermost layer of the skin and the body's primary barrier to external pathogens; however, the early epidermal immune response remains to be mechanistically understood. We show that the chemokine CXCL14, produced by epidermal keratinocytes, exhibits robust circadian fluctuations and initiates innate immunity. Clearance of the skin pathogen Staphylococcus aureus in nocturnal mice was associated with CXCL14 expression, which was high during subjective daytime and low at night. In contrast, in marmosets, a diurnal primate, circadian CXCL14 expression was reversed. Rhythmically expressed CXCL14 binds to S. aureus DNA and induces inflammatory cytokine production by activating Toll-like receptor (TLR)9-dependent innate pathways in dendritic cells and macrophages underneath the epidermis. CXCL14 also promoted phagocytosis by macrophages in a TLR9-independent manner. These data indicate that circadian production of the epidermal chemokine CXCL14 rhythmically suppresses skin bacterial proliferation in mammals by activating the innate immune system.

CNOT1 regulates circadian behaviour through Per2 mRNA decay in a deadenylation-dependent manner.

Circadian clocks are an endogenous internal timekeeping mechanism that drives the rhythmic expression of genes, controlling the 24 h oscillatory pattern in behaviour and physiology. It has been recently shown that post-transcriptional mechanisms are essential for controlling rhythmic gene expression. Controlling the stability of mRNA through poly(A) tail length modulation is one such mechanism. In this study, we show that Cnot1, encoding the scaffold protein of the CCR4-NOT deadenylase complex, is highly expressed in the suprachiasmatic nucleus, the master timekeeper. CNOT1 deficiency in mice results in circadian period lengthening and alterations in the mRNA and protein expression patterns of various clock genes, mainly Per2. Per2 mRNA exhibited a longer poly(A) tail and increased mRNA stability in Cnot1+/- mice. CNOT1 is recruited to Per2 mRNA through BRF1 (ZFP36L1), which itself oscillates in antiphase with Per2 mRNA. Upon Brf1 knockdown, Per2 mRNA is stabilized leading to increased PER2 expression levels. This suggests that CNOT1 plays a role in tuning and regulating the mammalian circadian clock.

Gpr19 is a circadian clock-controlled orphan GPCR with a role in modulating free-running period and light resetting capacity of the circadian clock.

Gpr19 encodes an evolutionarily conserved orphan G-protein-coupled receptor (GPCR) with currently no established physiological role in vivo. We characterized Gpr19 expression in the suprachiasmatic nucleus (SCN), the locus of the master circadian clock in the brain, and determined its role in the context of the circadian rhythm regulation. We found that Gpr19 is mainly expressed in the dorsal part of the SCN, with its expression fluctuating in a circadian fashion. A conserved cAMP-responsive element in the Gpr19 promoter was able to produce circadian transcription in the SCN. Gpr19-/- mice exhibited a prolonged circadian period and a delayed initiation of daily locomotor activity. Gpr19 deficiency caused the downregulation of several genes that normally peak during the night, including Bmal1 and Gpr176. In response to light exposure at night, Gpr19-/- mice had a reduced capacity for light-induced phase-delays, but not for phase-advances. This defect was accompanied by reduced response of c-Fos expression in the dorsal region of the SCN, while apparently normal in the ventral area of the SCN, in Gpr19-/- mice. Thus, our data demonstrate that Gpr19 is an SCN-enriched orphan GPCR with a distinct role in circadian regulation and may provide a potential target option for modulating the circadian clock.

Non-coding cis-element of Period2 is essential for maintaining organismal circadian behaviour and body temperature rhythmicity.

Non-coding cis-regulatory elements are essential determinants of development, but their exact impacts on behavior and physiology in adults remain elusive. Cis-element-based transcriptional regulation is believed to be crucial for generating circadian rhythms in behavior and physiology. However, genetic evidence supporting this model is based on mutations in the protein-coding sequences of clock genes. Here, we report generation of mutant mice carrying a mutation only at the E'-box cis-element in the promoter region of the core clock gene Per2. The Per2 E'-box mutation abolishes sustainable molecular clock oscillations and renders circadian locomotor activity and body temperature rhythms unstable. Without the E'-box, Per2 messenger RNA and protein expression remain at mid-to-high levels. Our work delineates the Per2 E'-box as a critical nodal element for keeping sustainable cell-autonomous circadian oscillation and reveals the extent of the impact of the non-coding cis-element in daily maintenance of animal locomotor activity and body temperature rhythmicity.

Calcitonin receptors are ancient modulators for rhythms of preferential temperature in insects and body temperature in mammals.

Daily body temperature rhythm (BTR) is essential for maintaining homeostasis. BTR is regulated separately from locomotor activity rhythms, but its molecular basis is largely unknown. While mammals internally regulate BTR, ectotherms, including Drosophila, exhibit temperature preference rhythm (TPR) behavior to regulate BTR. Here, we demonstrate that the diuretic hormone 31 receptor (DH31R) mediates TPR during the active phase in Drosophila DH31R is expressed in clock cells, and its ligand, DH31, acts on clock cells to regulate TPR during the active phase. Surprisingly, the mouse homolog of DH31R, calcitonin receptor (Calcr), is expressed in the suprachiasmatic nucleus (SCN) and mediates body temperature fluctuations during the active phase in mice. Importantly, DH31R and Calcr are not required for coordinating locomotor activity rhythms. Our results represent the first molecular evidence that BTR is regulated distinctly from locomotor activity rhythms and show that DH31R/Calcr is an ancient specific mediator of BTR during the active phase in organisms ranging from ectotherms to endotherms.

Circadian PER2 protein oscillations do not persist in cycloheximide-treated mouse embryonic fibroblasts in culture.

It is not known whether the endogenous mammalian core clock proteins sustain measurable oscillations in cells in culture where de novo translation is pharmacologically inhibited. We studied here the mammalian core clock protein PER2, which undergoes robust circadian oscillations in both abundance and phosphorylation. With a newly developed antibody that enables tracing the endogenous PER2 protein oscillations over circadian cycles with cultured mouse embryonic fibroblast cells, we provide evidence that PER2 does not persist noticeable circadian rhythms when translation is inhibited.

G-protein-coupled receptor signaling through Gpr176, Gz, and RGS16 tunes time in the center of the circadian clock [Review].

G-protein-coupled receptors (GPCRs) constitute an immensely important class of drug targets with diverse clinical applications. There are still more than 120 orphan GPCRs whose cognate ligands and physiological functions are not known. A set of circadian pacemaker neurons that governs daily rhythms in behavior and physiology resides in the suprachiasmatic nucleus (SCN) in the brain. Malfunction of the circadian clock has been linked to a multitude of diseases, such as sleeping disorders, obesity, diabetes, cardiovascular diseases, and cancer, which makes the clock an attractive target for drug development. Here, we review a recently identified role of Gpr176 in the SCN. Gpr176 is an SCN-enriched orphan GPCR that sets the pace of the circadian clock in the SCN. Even without known ligand, this orphan receptor has an agonist-independent basal activity to reduce cAMP signaling. A unique cAMP-repressing G-protein subclass Gz is required for the activity of Gpr176. We also provide an overview on the circadian regulation of G-protein signaling, with an emphasis on a role for the regulator of G-protein signaling 16 (RGS16). RGS16 is indispensable for the circadian regulation of cAMP in the SCN. Developing drugs that target the SCN remains an unfulfilled opportunity for the circadian pharmacology. This review argues for the potential impact of focusing on GPCRs in the SCN for the purpose of tuning the body clock.

Gpr176 is a Gz-linked orphan G-protein-coupled receptor that sets the pace of circadian behaviour.

G-protein-coupled receptors (GPCRs) participate in a broad range of physiological functions. A priority for fundamental and clinical research, therefore, is to decipher the function of over 140 remaining orphan GPCRs. The suprachiasmatic nucleus (SCN), the brain's circadian pacemaker, governs daily rhythms in behaviour and physiology. Here we launch the SCN orphan GPCR project to (i) search for murine orphan GPCRs with enriched expression in the SCN, (ii) generate mutant animals deficient in candidate GPCRs, and (iii) analyse the impact on circadian rhythms. We thereby identify Gpr176 as an SCN-enriched orphan GPCR that sets the pace of circadian behaviour. Gpr176 is expressed in a circadian manner by SCN neurons, and molecular characterization reveals that it represses cAMP signalling in an agonist-independent manner. Gpr176 acts independently of, and in parallel to, the Vipr2 GPCR, not through the canonical Gi, but via the unique G-protein subclass Gz.

Angiotensin II triggers expression of the adrenal gland zona glomerulosa-specific 3ß-hydroxysteroid dehydrogenase isoenzyme through de novo protein synthesis of the orphan nuclear receptors NGFIB and NURR1.

The 3ß-hydroxysteroid dehydrogenase (3ß-HSD) is an enzyme crucial for steroid synthesis. Two different 3ß-HSD isoforms exist in humans. Classically, HSD3B2 was considered the principal isoform present in the adrenal. However, we recently showed that the alternative isoform, HSD3B1, is expressed specifically within the adrenal zona glomerulosa (ZG), where aldosterone is produced, raising the question of why this isozyme needs to be expressed in this cell type. Here we show that in both human and mouse, expression of the ZG isoform 3ß-HSD is rapidly induced upon angiotensin II (AngII) stimulation. AngII is the key peptide hormone regulating the capacity of aldosterone synthesis. Using the human adrenocortical H295R cells as a model system, we show that the ZG isoform HSD3B1 differs from HSD3B2 in the ability to respond to AngII. Mechanistically, the induction of HSD3B1 involves de novo protein synthesis of the nuclear orphan receptors NGFIB and NURR1. The HSD3B1 promoter contains a functional NGFIB/NURR1-responsive element to which these proteins bind in response to AngII. Knockdown of these proteins and overexpression of a dominant negative NGFIB both reduce the AngII responsiveness of HSD3B1. Thus, the AngII-NGFIB/NURR1 pathway controls HSD3B1. Our work reveals HSD3B1 as a new regulatory target of AngII.

Immunolocalization of murine type VI 3ß-hydroxysteroid dehydrogenase in the adrenal gland, testis, skin, and placenta.

The enzyme 3ß-hydroxysteroid dehydrogenase/isomerase (3ß-HSD) is essential for the biosynthesis of all active steroid hormones, such as those secreted from the adrenal gland, testis, ovary, skin and placenta. The 3ß-HSD enzymes exist in multiple isoforms in humans and rodents. To date, six different isoforms have been identified in the mouse, and these isoforms are speculated to play different roles in different tissues. We previously showed that the murine type VI 3ß-HSD isoform (Hsd3b6) is expressed specifically in the aldosterone-producing zona glomerulosa cells within the adrenal gland and that its overexpression causes abnormally increased aldosterone synthesis, revealing a crucial (or rate-limiting) role of this enzyme in steroidogenesis. However, potential contributions of this enzyme to the steroid hormone synthesis outside the adrenal glands are poorly understood. This paucity of knowledge is partly because of the lack of isoform-specific antibody that can be used for immunohistochemistry. Here, we report the development and characterization of specific antibody to Hsd3b6 and show the results of immunohistochemistry for the adrenal gland, testis, ovary, skin and placenta. As expected, Hsd3b6 immunoreactivities within the adrenal gland were essentially confined to the zona glomerulosa cells, where aldosterone is produced. By contrast, no immunopositive cells were observed in the zona fasciculata, which is where corticosterone is produced. In the gonads, while the ovaries did not show any detectable immunoreactivity to Hsd3b6, the testes displayed intense immunoreactivities within the interstitial Leydig cells, where testosterone is produced. In the skin, positive immunoreactivities to Hsd3b6 were only seen in the sebaceous glands, suggesting a specific role of this enzyme in sebaceous function. Moreover, in the placenta, Hsd3b6 was specifically found in the giant trophoblast cells surrounding the embryonic cavity, which suggests a role for this enzyme in local progesterone production that is required for proper embryonic implantation and/or maintenance of pregnancy. Taken together, our data revealed that Hsd3b6 is localized in multiple specific tissues and cell types, perhaps thereby involved in biosynthesis of a number of tissue-specific steroid hormones with different physiological roles.

Effects of high potassium chloride supplementation on water intake and bodyweight gains in pregnant and lactating mice.

Thirty-one ICR pregnant mice were assigned to a control or a potassium chloride (KCl) diet group to clarify the effects of KCl supplementation on water intake, bodyweight gains and serum components in pregnant and lactating mice, and 5% KCl was supplemented in KCl diets from 6.5 days post coitus to 1 or 14 days after parturition. Feed intake was not affected by treatment, but supplemental KCl decreased bodyweight gains of lactating mice and their neonatal mice. Water intake and urine volume of KCl supplemented mice were significantly higher than those of control mice during pregnancy and supplemental KCl decreased serum urea N in pregnant mice. Supplemental KCl increased water intake drastically in lactating mice immediately after parturition and increased serum K at 14 days after parturition. Histological alteration using hematoxylin-eosin was not found in the kidney of each mouse at 1 or 14 days after parturition. These results indicate that high KCl supplementation accelerates water intake in lactating mice and prevents bodyweight gains of maternal and neonatal mice during lactation.

Relationships between urine pH and electrolyte status in cows fed forages.

Data of 20 balance measurements from Holstein dairy cows and urine samples from 24 Japanese Black beef cows were collected to evaluate the relationships between urine pH and electrolyte status in cows fed forages. The ratio of forages in the diet was 70-100% in dairy cows and beef cows were fed Italian ryegrass silage and wheat bran. Mean urine pH in dairy cows was 8.10, ranging from 7.27 to 8.71, and that in beef cows was 7.73, ranging from 7.42 to 8.12. There were positive correlations between urine pH and urinary K contents (P = 0.0012) or K intake (P = 0.019) in dairy cows, although plasma Na, Cl and K had no effect on urine pH. There was a weak negative correlation (P = 0.039) between urine pH and urinary Na content in dairy cows. However, there were no significant correlations between urine pH and urinary Na, Cl and K contents in beef cows. These results indicate that the concentrated urinary K due to the increased K intake may directly enhance urine pH in dairy cows fed mainly forages.

Effects of high potassium chloride supplementation on water intake, urine volume and nitrogen balance in mice.

Sixteen ICR male mice were assigned to a control diet group or a KCl diet group in metabolic cages to clarify the effects of KCl supplementation on water intake, urine volume and N balance, and 5% of KCl was supplemented in KCl diets for 4 or 8 weeks. Bodyweight of KCl supplemented mice was significantly higher than that of control mice from 24 to 28 days after treatment. Feed intake, water intake and urine volume of KCl supplemented mice were significantly higher than those of control mice, and the increased water intake and urine volume in KCl supplemented mice were 4.49 and 4.15 g, respectively. Urinary N, K and Cl excretion were significantly higher in KCl supplemented mice. Although N retention was not significantly different between control and KCl supplemented mice, N retention in KCl supplemented mice tended to be lower. Serum creatinine concentration at 8 weeks after treatment was lower in KCl supplemented mice. Histological alteration using hematoxylin-eosin and Sirius red staining was not found in the kidney of each mouse at 4 and 8 weeks after treatment. These results suggest that high KCl supplementation increases water intake, urine volume and urinary N excretion in mice.