Avian Melatonin Synthesis: Photic and Circadian Regulation of Serotonin N-Acetyltransferase mRNA in the Chicken Pineal Gland and Retina

Abstract: The circadian rhythms in melatonin production in the chicken pineal gland and retina reflect changes in the activity of serotonin N -acetyltransferase (arylalkylamine N -acetyltransferase; AA-NAT; EC 2.3.1.87). Here we determined that the chicken AA-NAT mRNA is detectable in follicular pineal cells and retinal photoreceptors and that it exhibits a circadian rhythm, with peak levels at night. AA-NAT mRNA was not detected in other tissues. The AA-NAT mRNA rhythm in the pineal gland and retina persists in constant darkness (DD) and constant lighting (LL). The amplitude of the pineal mRNA rhythm is not decreased in LL. Light appears to influence the phase of the clock driving the rhythm in pineal AA-NAT mRNA in two ways: The peak is delayed by ∼6 h in LL, and it is advanced by >4 h by a 6-h light pulse late in subjective night in DD. Nocturnal AA-NAT mRNA levels do not change during a 20-min exposure to light, whereas this treatment dramatically decreases AA-NAT activity. These observations suggest that the rhythmic changes in chicken pineal AA-NAT activity reflect, at least in part, clock-generated changes in mRNA levels. In contrast, changes in mRNA content are not involved in the rapid light-induced decrease in AA-NAT activity.     

Neural adrenergic/cyclic AMP regulation of the immunoglobulin E receptor alpha-subunit expression in the mammalian pinealocyte: a neuroendocrine/immune response link?

The high affinity immunoglobulin E receptor (FcepsilonRI) complex is dedicated to immunoglobulin E-mediated allergic responses. Expression of the FcepsilonRI receptor is thought to be relatively stable and limited to mast cells, basophils, eosinophils, monocytes, Langerhans cells, platelets, and neutrophils. We now report that the FcepsilonRIalpha and FcepsilonRIgamma polypeptides are expressed in the pinealocyte, the melatonin-secreting cell of the pineal gland. Moreover, Fcer1a mRNA levels increased approximately 100-fold at night to levels that were higher than in other tissues examined. Pineal FcepsilonRIalpha protein also increased markedly at night from nearly undetectable daytime levels. Our studies indicate that pineal Fcer1a mRNA levels are controlled by a well described neural pathway that controls pineal function. This pathway includes the master circadian oscillator in the suprachiasmatic nucleus and passes through central and peripheral structures. The circadian expression of FcepsilonRIalpha in the pineal gland is driven by this neural circuit via an adrenergic/cyclic AMP mechanism. Pineal FcepsilonRIalpha and FcepsilonRIgamma may represent a previously unrealized molecular link between the neuroendocrine and immune systems.     

Circadian and photic regulation of MAP kinase by Ras- and protein phosphatase-dependent pathways in the chick pineal gland

Chick pineal mitogen-activated protein kinase (MAPK) exhibits circadian activation and light-dependent deactivation at nighttime. Here we report that, in the chick pineal gland, levels of active forms of MAPK, MEK, Raf-1 and Ras exhibited synchronous circadian rhythms with peaks during the subjective night, suggesting a sequential activation of components in the classical Ras-MAPK pathway in a circadian manner. In contrast, the light-dependent deactivation of MAPK was not accompanied by any change of MEK activity, but it was attributed to the light-dependent activation of protein phosphatase dephosphorylating MAPK. These results indicate that the photic and clock signals regulate MAPK activity via independent pathways, and suggest a pivotal role of MAPK in photic entrainment and maintenance of the circadian oscillation.     

Molecular cloning, mRNA expression, and immunocytochemical localization of a putative blue-light photoreceptor CRY4 in the chicken pineal gland

In non-mammalian vertebrates, the pineal gland contains an endogenous circadian oscillator and serves as a photosensitive neuroendocrinal organ. To better understand the pineal phototransduction mechanism, we focused on the chicken putative blue-light photoreceptive molecule, Cryptochrome4 (cCRY4). Here we report the molecular cloning of pineal cCry4 cDNA, the in vivo expression of cCry4 mRNA, and the detection of cCRY4 protein. cCry4 is transcribed in a wide variety of chick tissues out of which the pineal gland and retina contain high levels of cCry4 mRNA. In the pineal gland, under 12 h light : 12 h dark cycles, the levels of both cCry4 mRNA and cCRY4 protein showed diurnal changes, and in cultured chick pineal cells, the cCry4 mRNA level was not only up-regulated by light but also controlled by circadian signals. Immunoblot analysis with a cCRY4-specific antibody detected cCRY4 in a soluble fraction of the pineal lysate. Immunocytochemistry revealed that cCRY4 was expressed in many parenchymal cells and a limited number of stromal cells. These cCRY4 features strikingly contrast with those of the chick pineal photoreceptor pinopsin, suggesting a possible temporal and/or spatial duplicity of the pineal photoreceptive system, the opsin- and CRY-based mechanisms.     

Methionine adenosyltransferase:adrenergic-cAMP mechanism regulates a daily rhythm in pineal expression

(S)-adenosylmethionine (SAM) is a critical element of melatonin synthesis as the methyl donor in the last step of the pathway, the O-methylation of N-acetyl 5-hydroxytryptamine by hydroxyindole-O-methyltransferase. The activity of the enzyme that synthesizes SAM, methionine adenosyltransferase (MAT), increases 2.5-fold at night in the pineal gland. In this study, we found that pineal MAT2A mRNA and the protein it encodes, MAT II, also increase at night, suggesting that the increase in MAT activity is caused by an increase in MAT II gene products. The night levels of MAT2A mRNA in the pineal gland were severalfold higher than in other neural and non-neural tissues examined, consistent with the requirement for SAM in melatonin synthesis. Related studies indicate that the nocturnal increase in MAT2A mRNA is caused by activation of a well described neural pathway that mediates photoneural-circadian regulation of the pineal gland. MAT2A mRNA and MAT II protein were increased in organ culture by treatment with norepinephrine (NE), the sympathetic neurotransmitter that stimulates the pineal gland at night. NE is known to markedly elevate pineal cAMP, and here it was found that cAMP agonists elevate MAT2A mRNA levels by increasing MAT2A mRNA synthesis and that drugs that block cAMP activation of cAMP dependent protein kinase block effects of NE. Therefore, the NE-cAMP dependent increase in pineal MAT activity seems to reflect an increase in MAT II protein, which occurs in response to cAMP-->protein kinase-dependent increased MAT2A expression. The existence of this MAT regulatory system underscores the importance that MAT plays in melatonin biogenesis. These studies also point to the possibility that SAM production in other tissues might be regulated through cAMP.     

大鼠松果体Clock基因和芳烷脘N-乙酰基转移酶基因的昼夜节律性表达及光照影响

探讨12h光照、12h黑暗交替(12h-light：12h-dark cycle,LD)及持续黑暗(constant darkness,DD)光制下松果体Clock基因和芳烷脘N-乙酰基转移酶基因(arylalkylamine N-acetyltransferase gene,NAT)是否存在昼夜节律性表达及其光反应变化。Sprague-Dawley大鼠在LD和DD光制下分别被饲养4周(n=36)和8周(n=36)后,在一昼夜内每隔4h采集一组松果体组织(n=6),提取总RNA,用竞争性定量RT-PCR测定不同昼夜时点样品中Clock及NAT基因的mRNA相对表达量,通过余弦法和ClockLab软件获取节律参数,并经振幅检验是否存在昼夜节律。结果如下：(1)在DD或LD光制下,松果体Clock和NAT基因mRNA的表达均呈现夜高昼低的节律性振荡(P＜0．05)。(2)与DD光制下比较,LD光制下松果体Clock和NAT基因的表达振幅及峰值相的mRNA水平均降低(P＜0．05)。(3)在DD或LD光制下,Clock和NAT基因之间显示相似的节律性表达(P＞0．05)。结果表明,Clock和NAT基因在松果体中存在同步的内源性昼夜节律表达,光照作用可使其表达下调。     

Characterization of oligopeptide transporter (PepT1) in grass carp (Ctenopharyngodon idella)

The oligopeptide transporter (PepT1) is located on the brush-border membrane of the intestinal epithelium, and plays an important role in dipeptide and tripeptide absorptions from protein digestion. In this study, we cloned the PepT1 cDNA from grass carp and characterized its expression profile in response to dietary protein and feed additives (sodium butyrate) treatments. The PepT1 gene encodes a protein of 714 amino acids with high sequence similarity with other vertebrate homologues. Expression analysis revealed highest levels of PepT1 mRNA expression in the foregut of grass carp. In addition, PepT1 mRNA expression exhibited diurnal variation in all three bowel segments of intestine with lower levels of expression in daytime than nighttime. During embryonic development, PepT1 showed a dynamic pattern of expression reaching maximal levels of expression in the gastrula stage and minimal levels in the organ stage. The PepT1 expression showed constant levels from 14 to 34 day post-hatch. To determine whether fish diet of different protein contents may have any effect on PepT1 expression, we extended our research to dietary regulation of PepT1 expression. We found that dietary protein levels had a significant effect on PepT1 gene expression. In addition, PepT1 mRNA levels were higher after feeding with fish meal than with soybean meal. Moreover, in vitro and in vivo sodium butyrate treatments increased PepT1 expression in the intestine of grass carp. The results demonstrate for the first time that PepT1 mRNA expression is regulated in a temporal and spatial pattern during development, and dietary protein and feed additives had a significant effects on PepT1 gene expression in grass carp.     

Systematic Identification of Rhythmic Genes Reveals camk1gb as a New Element in the Circadian Clockwork

A wide variety of biochemical, physiological, and molecular processes are known to have daily rhythms driven by an endogenous circadian clock. While extensive research has greatly improved our understanding of the molecular mechanisms that constitute the circadian clock, the links between this clock and dependent processes have remained elusive. To address this gap in our knowledge, we have used RNA sequencing (RNA–seq) and DNA microarrays to systematically identify clock-controlled genes in the zebrafish pineal gland. In addition to a comprehensive view of the expression pattern of known clock components within this master clock tissue, this approach has revealed novel potential elements of the circadian timing system. We have implicated one rhythmically expressed gene, camk1gb, in connecting the clock with downstream physiology of the pineal gland. Remarkably, knockdown of camk1gb disrupts locomotor activity in the whole larva, even though it is predominantly expressed within the pineal gland. Therefore, it appears that camk1gb plays a role in linking the pineal master clock with the periphery.     

Lcg is a light-inducible and clock-controlled gene expressed in the chicken pineal gland

The circadian clock is an autonomous biological clock that is entrainable to environmental 24-h cycles by receiving time cues such as light. Generally, light given at early and late subjective night, respectively, delays and advances the phase of the circadian oscillator. We previously searched for the chicken pineal genes that are induced by light in a phase-dependent manner. The present study undertook cDNA cloning and characterization of a gene whose expression was remarkably up-regulated by light at late subjective night. The mRNA level of this gene exhibited robust diurnal change in the pineal gland, with a peak in the early (subjective) day under light-dark cycles and constant dark condition, and hence it was designated Lcg (Light-inducible and Clock-controlled Gene). Chicken Lcg encodes a coiled-coil protein composed of 560 amino acid residues. Among chicken tissues, the pineal gland and the retina exhibited relatively high expression levels of LCG. LCG was colocalized with gamma-tubulin, a centrosomal protein, when expressed in COS7 cells, and LCG is the first example of a clock-related molecule being accumulated at the centrosome. Coimmunoprecipitation of LCG with gamma-tubulin in the chicken pineal lysate suggests a link between the circadian oscillator and the centrosomal function.     

MicroRNA-193a-3p Reduces Intestinal Inflammation in Response to Microbiota via Down-regulation of Colonic PepT1

Intestinal inflammation is characterized by epithelial disruption, leading to the loss of barrier function, recruitment of immune cells, and host immune responses to gut microbiota. PepT1, a di/tripeptide transporter that uptakes bacterial products, is up-regulated in inflamed colon tissue, which implies its role in bacterium-associated intestinal inflammation. Although microRNA (miRNA)-mediated gene regulation has been found to be involved in various processes of inflammatory bowel disease (IBD), the biological function of miRNAs in the pathogenesis of IBD remains to be explored. In this study we detected miRNA expression patterns in colon tissues during colitis and investigated the mechanism underlying the regulation of colonic PepT1 by miRNAs. We observed an inverse correlation between PepT1 and miR-193a-3p in inflamed colon tissues with active ulcerative colitis, and we further demonstrated that miR-193a-3p reduced PepT1 expression and activity as a target gene and subsequently suppressed the NF-κB pathway. Intracolonic delivery of miR-193a-3p significantly ameliorated dextran sodium sulfate-induced colitis, whereas the overexpression of colonic PepT1 via PepT1 3′-untranslated region mutant lentivirus vector abolished the anti-inflammatory effect of miR-193a-3p. Furthermore, antibiotic treatment eliminated the difference in the dextran sodium sulfate-induced inflammation between the presence and absence of miR-193a-3p. These findings suggest that miR-193a-3p regulation of PepT1 mediates the uptake of bacterial products and is a potent mechanism during the colonic inflammation process. Overall, we believe miR-193a-3p may be a potent regulator of colonic PepT1 for maintaining intestinal homeostasis.     

松果体昼夜节律生物钟分子机制的研究进展

在各种非哺乳类脊椎动物中 ,松果体起着中枢昼夜节律振荡器的作用。近来 ,在鸟类松果体中相继发现了几种钟基因 ,如Per、Cry、Clock和Bmal等 ,其表达的时间变化规律与哺乳类视交叉上核 (SCN)的非常相似。钟的振荡由其自身调控反馈环路的转录和翻译组成 ,鸟类松果体和哺乳类SCN似乎具有共同的钟振荡基本分子构架 ;若干钟基因产物作为正向或负向调节子影响钟的振荡 ;昼夜性的控时机制同时也需要翻译后事件的参与。这些过程对钟振荡器的稳定性和 /或钟导引的光输入通路有着重要的调控作用