Clock gene expression in the submandibular glands

Clock genes, which mediate molecular circadian rhythms, are expressed in a circadian fashion in the suprachiasmatic nucleus and in various peripheral tissues. To establish a molecular basis for circadian regulation in the salivary glands, we examined expression profiles of clock-related genes and salivary gland-characteristic genes. Clock-related genes-including Per1, Per2, Cry1, Bmal1, Dec1, Dec2, Dbp, and Reverbalpha-showed robust circadian expression rhythms in the submandibular glands in 12:12-hour light-dark conditions. In addition, a robust circadian rhythm was observed in amylase 1 mRNA levels, whereas the expression of other salivary-gland-characteristic genes examined was not rhythmic. The Clock mutation resulted in increased or decreased mRNA levels of Per2, Bmal1, Dec1, Dec2, and Dbp, and in Cry1-/- background, Cry2 disruption also increased or decreased mRNA levels of these clock-related genes and the amylase 1 gene. These findings indicate that the Clock- and Cry-dependent molecular clock system is active in the salivary glands.     

Genetic regulation of salivary proteins in rodents

The presence of a protein in the cell is the result of a complex pathway that is known by the term gene expression. In this article we review the existing literature on the structure and expression of representative salivary gland genes and their regulated expression during development and upon extracellular stimulation. The expression of one of the "nuclear" protooncogenes, c-fos, in rat parotid glands is also discussed. Finally, we present some suggestions for future studies that will help to understand the mechanisms leading to gene regulation in rat salivary glands.     

A mouse caries model and evaluation of aqp5-/- knockout mice

Current techniques to alter gene expression in mice allow direct analysis of the net role of a host factor in caries development. Towards this goal we first established protocols to induce and score caries in NFS/N mice and determined caries susceptibility in mice with targeted deletion of the gene encoding aquaporin-5 (Aqp5-/-), a water channel involved in the production of saliva. In the NFS/N strain of mice total sulcal caries and severity scores were consistent between experiments, whereas smooth surface caries scores were lower, more variable but distributed fairly evenly among the buccal, lingual and sulcal surfaces. In Black Swiss/129SvJ mice (genetic background of Aqp5-/- mice) caries scores were 50-75% lower compared to NFS/N mice, suggesting strain variation in caries susceptibility under our experimental conditions. In Aqp5-/- mice, in which the volume of total salivary secretion is reduced by 60-65%, there was a significant increase in caries, primarily on the buccal and sulcal surfaces. Results indicate that caries susceptibility increases with a reduced salivary flow that is associated with decreased water content of saliva.     

钟基因Per2和肿瘤相关钟控基因在金黄地鼠口腔颊黏膜癌变不同阶段的昼夜节律改变

目的 探讨钟基因Per2和钟控基因血管内皮生长因子（VEGF）、Ki67、c-Myc和P53在颊黏膜癌变不同阶段的昼夜节律变化规律以及与癌变发生发展的关系。方法 90只叙利亚金黄地鼠置于12 h光照和12 h黑暗交替环境中饲养,用二甲基苯并蒽（DMBA）涂抹颊黏膜建立金黄地鼠颊癌模型,分别在DMBA涂抹前,涂抹6周和14周后的24 h内的6个不同时间点处死动物,获取正常颊黏膜、癌前病变和癌症3个不同阶段昼夜6个不同时间的组织。经病理学检查确认后,采用实时荧光定量聚合酶链反应检测各时间点Per2、VEGF、Ki67、c-Myc和P53 mRNA的相对表达量,并行余弦分析,以中值、振幅和峰值位相时为指标反映各基因表达的昼夜节律特征。结果 Per2、VEGF、P53和c-Myc mRNA在癌变3个阶段的表达均具有昼夜节律性（P<0.05）,而Ki67 mRNA仅在正常黏膜和癌前病变阶段的表达具有昼夜节律性（P<0.05）。Per2和P53 mRNA表达的中值随着癌症的发展而降低（P<0.05）,VEGF、c-Myc和Ki67 mRNA表达的中值随着癌症的发展而上升（P<0.05）;P53 mRNA的振幅随着癌症的发展而降低（P<0.05）,Per2、VEGF、Ki67和c-Myc mRNA的振幅在癌前病变和癌症阶段均高于正常组（P<0.05）;在癌前病变阶段,Per2、VEGF和c-Myc mRNA的峰值位相时较正常组提前,而Ki67和P53 mRNA的峰值位相时较正常组滞后。结论 随着癌症的发生和发展,钟基因Per2和肿瘤相关钟控基因VEGF、Ki67、c-Myc、P53表达的昼夜节律特征发生明显改变。     

Review of genes potentially related to hyposecretion in male non-obese diabetic (NOD) mice,a Sjögren's syndrome model

BackgroundSjögren's syndrome (SS) is known to cause dry eyes and mouth due to inflammation of the lacrimal and salivary glands. However, some reports imply that other factors trigger dry eyes and mouth. We previously investigated various factors using RNA-sequencing analysis of lacrimal glands from male non-obese diabetic (NOD) mice, an SS model. In this review, we described (1) the exocrine features of male and female NOD mice, (2) the up- and down-regulated genes in the lacrimal glands of male NOD mice as revealed by our RNA-sequencing data, and (3) comparisons between these genes and data in the Salivary Gland Gene Expression Atlas.HighlightsMale NOD mice exhibit a steady worsening of lacrimal hyposecretion and dacryoadenitis, whereas females exhibit a complex pathophysiological condition that includes diabetic disease, salivary hyposecretion, and sialadenitis. Ctss, an up-regulated gene, is a potential inducer of lacrimal hyposecretion and is also expressed in salivary glands. Two other up-regulated genes, Ccl5 and Cxcl13, may worsen the inflammation of SS in both the lacrimal and salivary glands. The genes Esp23, Obp1a, and Spc25 were detected as down-regulated, but judging the relationship between these genes and hyposecretion is difficult as only limited information is available. Another down-regulated gene, Arg1, is involved in lacrimal hyposecretion, and it also has the potential to cause salivary hyposecretion in NOD mice.ConclusionIn NOD mice, males may be better than females at evaluating the pathophysiology of SS. Some regulated genes revealed by our RNA-sequencing data might be potential therapeutic targets for SS.     

Sex-related differences in gene expression in salivary glands of BALB/c mice

Sex-related differences exist in the structure and function of the major glands in a variety of species. Moreover, many of these variations appear to be unique to each tissue. We hypothesized that this sexual dimorphism is due, at least in part, to gland-specific differences in gene expression between males and females. Glands were collected from male and female BALB/c mice (n = 5/sex/experiment), and total RNA was isolated. Samples were analyzed for differentially expressed mRNAs with CodeLink microarrays, and data were evaluated by GeneSifter. Our results demonstrate that significant (P < 0.05) sex-related differences exist in the expression of numerous genes in the major salivary glands, and many of these differences were tissue-specific. These findings support our hypothesis that sex-related differences in the salivary glands are due, at least in part, to tissue-specific variations in gene expression.     

Alterations of p16INK4a tumour suppressor gene in mucoepidermoid carcinoma of the salivary glands

Mucoepidermoid carcinoma (MEC) is common in the salivary glands, but alterations of the p16(INK4a) tumour suppressor gene are largely unknown. The aim of this study was to analyse p16(INK4a) gene alterations in MEC, and evaluate their significance for carcinogenesis. Thirty-eight salivary glands with MEC and six normal salivary glands were studied for p16(INK4a) alterations. In the MEC-affected group, there were 23.7% (9/38) and 13.2% (5/38) cases of homozygous deletion, and 5.3% (2/38) and 2.6% (1/38) cases of point mutation in p16(INK4a) exon 1 and exon 2, respectively. Hypermethylation of the p16(INK4a) gene promoter was found in 13 cases (13/38, 34.2%). Alterations of the p16(INK4a) gene were not found in the normal salivary glands. These findings suggest that the main mechanisms of inactivation of the p16(INK4a) gene in MEC of the salivary glands are promoter hypermethylation and homozygous deletion.     

An AAV2/5 vector enhances safety of gene transfer to the mouse salivary gland

This study was designed to improve AAV-mediated gene transfer to the murine submandibular salivary glands. Our first aim was to utilize AAV pseudotype vectors, containing the genetic elements of the canonical AAV2, packaged within capsids of AAV serotypes 5, 8, and 9. Having determined that this pseudotyping increased the efficiency of gene transfer to the glands by several orders of magnitude, we next asked whether we could reduce the gene transfer inoculum of the pseudotype while still achieving gene transfer comparable with that achieved with high-dose AAV2. Having achieved gene transfer comparable with that of AAV2 using a pseudotype vector (AAV2/5) at a 100-fold lower dose, our final objective was to evaluate the implications of this lower dose on two pre-clinical parameters of vector safety. To evaluate systemic toxicity, we measured AAV vector sequestration in the liver using qPCR, and found that the 100-fold lower dose reduced the vector recovered from the liver by 300-fold. To evaluate salivary gland function, we undertook whole-proteome profiling of salivary gland lysates two weeks after vector administration and found that high-dose (5 × 10?) AAV altered the expression level of ~32% of the entire salivary gland proteome, and that the lower dose (5 × 10?) reduced this effect to ~7%.     

Effect of photic stimuli on rat salivary glands. Role of sympathetic nervous system

Saliva secretion during feeding facilitates chewing, swallowing and other oral functions. Between meals, a "resting saliva" is elicited to allow speaking and contribute to maintain soft and hard tissues health. Chewing is the main stimulus for "stimulated saliva" secretion. Mouth dryness and other less well known stimuli control "resting saliva". In humans the stimulus of the light increases the parotid saliva flow rate. Saliva secretion occurs in response to a reflex. Both motor branches of the autonomous nervous system drive efferent outputs to the salivary glands. Cellular bodies of sympathetic motor fibers innervating salivary glands are located in the superior cervical ganglia. A multisynaptic pathway couples the superior cervical ganglia to hypothalamic areas related to the control of autonomous and endocrine functions. Projections from suprachiasmatic nuclei involved in circadian rhythms control reach those areas. Salivary glands postsynaptic beta-adrenoceptors control synthesis and secretion of proteins. Postsynaptic alpha 2-adrenoceptors modulate salivary responses mediated by alpha 1 and beta-adrenoceptors. Parotid alpha-amylase circadian rhythm in suckling rats, suggest that the sympathetic nervous system mediates an effect of light on saliva secretion. Analysis of: 1) parotid fine structure, 2) submandibular secretory response to adrenergic agonists, and 3) submandibular 3H-clonidine binding to alpha 2-adrenoceptors, demonstrated that an increase of sympathetic reflex activity occurs in salivary glands of rats chronically exposed to constant light. Similar effects were observed in rats chronically exposed to immobilization stress. Catecholamine biosynthetic enzyme mRNA levels in adrenal glands and superior cervical ganglia suggest that changes induced by light on salivary sympathetic reflex activity are mediated by plasma catecholamines released by adrenal glands. Post and presynaptic alpha 2 adrenoceptors could play an important role in saliva secretion control when light or stress stimuli modify the sympathoadrenal system.     

生物钟基因Per1对人口腔鳞状细胞癌细胞增殖、凋亡、细胞周期和体内成瘤的影响及机理

目的 探讨生物钟基因Per1对细胞周期相关基因的调控作用,以及对人口腔鳞状细胞癌细胞SCC15增殖、凋亡、细胞周期和体内成瘤的影响。方法 构建3组针对Per1 RNA的短发夹RNA（shRNA）慢病毒重组质粒,转染SCC15细胞,用蛋白质印迹法（Western blot）及实时荧光定量PCR（qRT-PCR）检测筛选RNA干扰作用最强组为实验组,对照组（Control-shRNA）为对任何基因均无干扰效应的shRNA序列的质粒,空白组为未做任何处理的SCC15细胞。用qRT-PCR检测各组细胞中细胞周期相关基因Per1、p53、Cyclin D1、Cyclin E、Cyclin A2、Cyclin B1、CDK1、CDK2、CDK4、CDK6、p16、p21、Wee1、cdc25、E2F、Rb1 mRNA的表达;用流式细胞仪检测各组细胞增殖、凋亡和细胞周期分布;将实验组和空白组细胞分别接种于裸鼠背部皮下,观察成瘤情况。结果 成功构建了3组Per1-shRNA慢病毒质粒,通过qRT-PCR和Western blot证明Per1-shRNA-Ⅰ组沉默效果最佳,作为实验组。Per1-shRNA-Ⅰ组中Cyclin D1、Cyclin E、Cyclin B1、CDK1和Wee1 mRNA的表达水平高于Control-shRNA组和SCC15组（P<0.05）,p53、Cyclin A2、p16、p21和cdc25 mRNA的表达水平降低（P<0.05）;Control-shRNA组和SCC15组中各基因mRNA的表达水平无差异（P>0.05）。CDK2、CDK4、CDK6、E2F和Rb1 mRNA的表达水平在3组中均无统计学差异（P>0.05）。Per1-shRNA-Ⅰ组中细胞增殖指数高于Control-shRNA组和SCC15组（P<0.05）,凋亡指数降低（P<0.05）,S期的细胞数降低（P<0.05）,G2/M期的细胞数增加（P<0.05）。Control-shRNA组和SCC15组细胞的增殖指数和凋亡指数无统计学差异（P>0.05）。Per1-shRNA-Ⅰ组细胞体内成瘤能力显著增强（P<0.05）。结论 生物钟基因Perl是重要的抑癌基因,Perl能调控下游众多的细胞周期基因,其表达变化影响细胞周期进程、增殖和凋亡的平衡失调及体内成瘤能力,对Per1深入研究有可能进一步明确癌症的发生发展机制,为癌症的治疗提供新的有效分子靶点。     

Viral gene transfer to developing mouse salivary glands

Branching morphogenesis is essential for the formation of salivary glands, kidneys, lungs, and many other organs during development, but the mechanisms underlying this process are not adequately understood. Microarray and other gene expression methods have been powerful approaches for identifying candidate genes that potentially regulate branching morphogenesis. However, functional validation of the proposed roles for these genes has been severely hampered by the absence of efficient techniques to genetically manipulate cells within embryonic organs. Using ex vivo cultured embryonic mouse submandibular glands (SMGs) as models to study branching morphogenesis, we have identified new vectors for viral gene transfer with high efficiency and cell-type specificity to developing SMGs. We screened adenovirus, lentivirus, and 11 types of adeno-associated viruses (AAV) for their ability to transduce embryonic day 12 or 13 SMGs. We identified two AAV types, AAV2 and bovine AAV (BAAV), that are selective in targeting expression differentially to SMG epithelial and mesenchymal cell populations, respectively. Transduction of SMG epithelia with self-complementary (sc) AAV2 expressing fibroblast growth factor 7 (Fgf7) supported gland survival and enhanced SMG branching morphogenesis. Our findings represent, to our knowledge, the first successful selective gene targeting to epithelial vs. mesenchymal cells in an organ undergoing branching morphogenesis.     

沉默生物钟基因PER1对口腔鳞状细胞癌细胞中生物钟基因网络的影响

目的 探讨口腔鳞状细胞癌细胞中生物钟基因PER1对生物钟基因网络中其他生物钟基因表达的影响。方法 采用RNA干扰技术沉默人口腔鳞状细胞癌SCC15细胞中PER1基因,应用流式细胞仪检测沉默后细胞的增殖和凋亡水平,实时荧光定量聚合酶链式反应检测生物钟基因CLOCK、BMAL1、PER1、PER2、PER3、DEC1、DEC2、CRY1、CRY2、TIM、CKIE、RORA、NPAS2、REV-ERBA mRNA的表达情况。结果 PER1基因沉默后,SCC15细胞增殖指数上升,凋亡指数下降（P<0.05）;PER1、PER2、DEC1、DEC2、CRY1、CRY2和NPAS2 mRNA的表达水平降低（P<0.05）,PER3、TIM、RORA和REV-ERBA mRNA的表达水平升高（P<0.05）,CLOCK、BMAL1和CKIE mRNA的表达水平无统计学改变（P>0.05）。结论 生物钟基因PER1能够调控生物钟基因网络中众多其他生物钟基因PER2、DEC1、DEC2、CRY1、CRY2、NPAS2、PER3、TIM、RORA和REV-ERBA的表达,PER1在生物钟基因网络中具有重要调控作用。     

Expression of podoplanin in the mouse salivary glands

OBJECTIVE: Podoplanin is one of the most highly expressed lymphatic-specific genes. Here, we report the distribution of cells expressing podoplanin in mouse salivary glands. DESIGN: We immunohistochemically investigated the distribution of cells expressing podoplanin in mouse major salivary glands by laser-scanning microscopy. The expression of endothelial cell marker PECAM-1 was tested to discriminate lymphatic endothelium from salivary gland cells, and myoepithelial cells were identified by an antibody for P-cadherin. RESULTS: The podoplanin expression was rarely found in acini of the parotid gland but clearly found at the basal portion of acini in the submandibular and sublingual glands. The number of portion reacted with anti-podoplanin is greater in the sublingual gland than in the submandibular gland. The expression was also found at the basal portion of ducts in all major salivary glands. The P-cadherin expression was rarely found in acini of the parotid gland but found in acini of the sublingual gland and on ducts in parotid and sublingual glands, corresponding to the area of podoplanin expression. CONCLUSIONS: It was suggested that the acinar and myoepithelial cells in the salivary glands have the ability to express podoplanin, and that the expression may be concerned with the mucous saliva excretion.