The hypothesis that L-glutamate (Glu) is an excitatory amino acid neurotransmitter in the mammalian central nervous system is now gaining more support after the successful cloning of a number of genes coding for the signaling machinery required for this neurocrine at synapses in the brain. These include Glu receptors (signal detection), Glu transporters (signal termination) and vesicular Glu transporters (signal output through exocytotic release). Relatively little attention has been paid to the functional expression of these molecules required for Glu signaling in peripheral neuronal and non-neuronal tissues; however, recent molecular biological analyses show a novel function for Glu as an extracellular signal mediator in the autocrine and/or paracrine system. Emerging evidence suggests that Glu could play a dual role in mechanisms underlying the maintenance of cellular homeostasis -as an excitatory neurotransmitter in the central neurocrine system and an extracellular signal mediator in peripheral autocrine and/or paracrine tissues. In this review, the possible Glu signaling methods are outlined in specific peripheral tissues including bone, testis, pancreas, and the adrenal, pituitary and pineal glands.Keywords: autocrine; glutamate; glutamate receptor; glutamate transporter; neurotransmitter; paracrine; vesicular glutamate transporter; peripheral tissues. Glutamate signaling moleculesGlutamate receptors L-Glutamate (Glu) is accepted as an excitatory amino acid neurotransmitter in the mammalian central nervous system (CNS). Receptors for Glu (GluRs) are categorized into two major classes, metabotropic (mGluRs) and ionotropic (iGluRs) receptors, according to their differential intracellular signal transduction mechanisms and molecular homologies (Fig. 1) [1-3]. mGluRs are further divided into three distinct subtypes containing seven transmembrane domains, including group I (mGluR1 and mGluR5), group II (mGluR2 and mGluR3) and group III (mGluR4, mGluR6, mGluR7 and mGluR8), in line with each receptor's exogenous agonists and intracellular second messengers [4,5]. The group I subtype stimulates formation of inositol 1,4,5-triphosphate and diacylglycerol, while both group II and III subtypes induce reduction of intracellular cyclic AMP (cAMP). On the basis of sequence homology and agonist preference, the latter iGluRs are classified into N-methyl-D-aspartate (NMDA), DL-a-amino-3-hydroxy-5-methylisoxasole-4-propionate (AMPA), and kainate (KA) receptors, which are associated with ion channels permeable to particular cations [6,7].NMDA receptor channels. These channels are highly permeable to Ca 2+ , with sensitivity to blockade by Mg 2+ in a voltage-dependent manner [8,9]. Functional NMDA receptor channels are comprised of heteromeric assemblies between the essential NR1 subunit and one of four different NR2 (A-D) subunits, in addition to one of two different NR3 (A-B) subunits. Expression of the NR2 subunit alone does not lead to composition of functional ion channels in any expression system, while coexpression of...

DEC1 suppresses CLOCK/BMAL1-enhanced promoter activity, but its role in the circadian system of mammals remains unclear. Here we examined the effect of Dec1 overexpression or deficiency on circadian gene expression triggered with 50% serum. Overexpression of Dec1 delayed the phase of clock genes such as Dec1, Dec2, Per1, and Dbp that contain E boxes in their regulatory regions, whereas it had little effect on the circadian phase of Per2 and Cry1 carrying CACGTT E boxes. In contrast, Dec1 deficiency advanced the phase of the E-box-containing clock genes but not that of the E-box-containing clock genes. Accordingly, DEC1 showed strong binding and transrepression on the E box, but not on the E box, in chromatin immunoprecipitation, electrophoretic mobility shift, and luciferase reporter assays. Dec1 ؊/؊ mice showed behavioral rhythms with slightly but significantly longer circadian periods under conditions of constant darkness and faster reentrainment to a 6-h phase-advanced shift of a light-dark cycle. Knockdown of Dec2 with small interfering RNA advanced the phase of Dec1 and Dbp expression, and double knockdown of Dec1 and Dec2 had much stronger effects on the expression of the E-box-containing clock genes. These findings suggest that DEC1, along with DEC2, plays a role in the finer regulation and robustness of the molecular clock.The mammalian molecular clock system consists of various clock genes and their protein products involved in interlocked feedback loops of transcriptional and translational regulation through clock elements such as CACGTG E-box, D-box, and ROR/REV-ERB binding elements (RORE) (18,24). Among these regulatory sequences, the E box is thought to be the most important element in the molecular oscillatory system, since it is the binding site for the CLOCK/BMAL1 heterodimer, which up-regulates various clock genes, including Dec1, Dec2, Per1, Dbp, and Rev-erb␣. In this regulatory system, PER, CRY, and DEC serve as negative factors for transcription from E-boxdriven promoters, and the E-box-like element EЈ box (CAC GTT) was recently shown to be involved in the direct regulation of Per2 and Cry1 genes by CLOCK/BMAL1 (1, 31, 34). RORE, on the other hand, is a clock element to which the transcriptional activators-ROR␣/ROR␤/ROR␥-and repressors-REV-ERB␣/REV-ERB␤-bind, and ROR and REV-ERB regulate the circadian expression of Bmal1, Clock, Npas2, and Cry1 via RORE (31).In the mammalian clock system, DEC1 (also known as BHLHB2, STRA13, or SHARP2) and DEC2 (BHLHB3 or SHARP1) serve as transcriptional repressors for CLOCK/ BMAL1-enhanced promoter activity, through binding to E boxes or interaction with BMAL1 (12,14,18,26). Among suppressive factors for E boxes, DEC1 and DEC2 can bind directly to E boxes through their basic helix-loop-helix DNA binding domains (18,26), although it remains unclear whether DEC1 and DEC2 also bind to the EЈ boxes. In contrast, PER and CRY interact with the CLOCK/BMAL1 heterodimer but cannot bind directly to E/EЈ boxes, since they have no DNA binding domain.Dec1 expression shows...

To elucidate the food-entrainable oscillatory mechanism of peripheral clock systems, we examined the effect of fasting on circadian expression of clock genes including Dec1 and Dec2 in mice. Withholding of food for 2 days had these effects: the expression level of Dec1 mRNA decreased in all tissues examined, although Per1 mRNA level markedly increased; Per2 expression was reduced in the liver and heart only 42-46 h after the start of fasting; and expression profiles of Dec2 and Bmal1 were altered only in the heart and in the liver, respectively, whereas Rev-erbalpha mRNA levels did not change significantly. Re-feeding after 36-h starvation erased, at least in part, the effect of fasting on Dec1, Dec2, Per1, Per2, and Bmal1 within several hours, and restriction feeding shifted the phase of expression profiles of all examined clock genes including Dec1 and Dec2. These findings indicate that short-term fasting and re-feeding modulate the circadian rhythms of clock genes to different extents in peripheral tissues, and suggest that the expression of Dec1, Per1, and some other clock genes was closely linked with the metabolic activity of these tissues.

In bone, clock genes are involved in the circadian oscillation of bone formation and extracellular matrix expression. However, to date little attention has been paid to circadian rhythm in association with expression of clock genes during chondrogenesis in cartilage. In this study, we investigated the functional expression of different clock genes by chondrocytes in the course of cartilage development. The mRNA expression of types I, II, and X collagens exhibited a 24-h rhythm with a peak at zeitgeber time 6, in addition to a 24-h rhythmicity of all the clock genes examined in mouse femurs in vivo. Marked expression of different clock genes was seen in both osteoblastic MC3T3-E1 and chondrogenic ATDC5 cells in vitro, whereas parathyroid hormone (PTH) transiently increased period 1 (per1) mRNA expression at 1 h in both cell lines. Similar increases were seen in the mRNA levels for both per1 and per2 in prehypertrophic chondrocytes in metatarsal organotypic cultures within 2 h of exposure to PTH. PTH significantly activated the mouse per1 (mper1) and mper2 promoters but not the mper3 promoter in a manner sensitive to both a protein kinase A inhibitor and deletion of the cAMP-responsive element sequence (CRE) in ATDC5 cells. In HEK293 cells, introduction of brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (bmal1)/clock enhanced mouse type II collagen first intron reporter activity without affecting promoter activity, with reduction effected by either per1 or per2. These results suggest that PTH directly stimulates mper expression through a protein kinase A-CRE-binding protein signaling pathway for subsequent regulation of bmal1/clock-dependent extracellular matrix expression in cartilage.Recent studies have revealed that the endogenous circadian rhythmicity generated at the cellular level by circadian core oscillators resides not only in the hypothalamic suprachiasmatic nucleus of the anterior hypothalamus (1, 2), which is recognized as the mammalian central clock, but also in various peripheral tissues including heart (3), adipose tissue (4), pancreas (5), and liver (2). The suprachiasmatic nucleus is not essential for driving peripheral oscillations but rather acts as a synchronizer of peripheral oscillators, whereas the physiological rhythmicity may be under the direct control by their own local clock genes in peripheral tissues (7). In mice, the rhythmic transcription of two orthologs of the Drosophila Period (per) gene appears to be essential for circadian rhythms. Expression of mouse per (mper) genes is known to be positively regulated by other clock proteins belonging to the basic helix-loop-helix period/aryl hydrocarbon receptor nuclear translocator/single minded class, which are Clock and brain and muscle aryl hydrocarbon receptor nuclear translocator-like protein 1 (Bmal1), 2 respectively. In addition, mPer proteins constitute multimeric complexes with products of the cryptochrome (cry) genes, mcry1 and mcry2, which in turn negatively regulate the gene transcription mediated ...

DEC1 (BHLHB2/Stra13/Sharp2)—a basic helix‐loop‐helix transcription factor—is known to be involved in various biological phenomena including clock systems and metabolism. In the clock systems, Dec1 expression is dominantly up‐regulated by CLOCK : BMAL1 heterodimer, and it exhibits circadian rhythm in the suprachiasmatic nucleus (SCN)—the central circadian pacemaker—and other peripheral tissues. Recent studies have shown that the strong circadian rhythmicity of Dec1 in the SCN was abolished by Clock mutation, whereas that in the liver was affected, but not abolished, by Clock mutation. Moreover, feeding conditions affected hepatic Dec1 expression, which indicates that Dec1 expression is closely linked with the metabolic functions of the liver. Among ligand‐activated nuclear receptors examined, LXRα and LXRβ with T0901317—agonist for LXR—were found to be potent enhancers for Dec1 promoter activity, and a higher expression level of LXRα protein was detected in the liver than in the kidney and heart. T0901317 increased the levels of endogenous Dec1 transcript in hepatoma cells. Chromatin immunoprecipitation assay indicated that LXRα bound to the Dec1 promoter, and an LXRα‐binding site was identified. These observations indicate that hepatic DEC1 mediates the ligand‐dependent LXR signal to regulate the expression of genes involved in the hepatic clock system and metabolism.