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, 11 (4), 32

β-Arrestin 1 (ARRB1) Serves as a Molecular Marker of the Peripheral Circadian Rhythm

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β-Arrestin 1 (ARRB1) Serves as a Molecular Marker of the Peripheral Circadian Rhythm

Tatsunosuke Tomita et al. Int J Oral Sci.

Abstract

The control of the circadian rhythm is important for health because it regulates physiological functions and is associated with health hazards. We aimed to identify a circadian biomarker of health status in human saliva, since collecting saliva is non-invasive, straightforward, and cost-effective. Among 500 genes potentially controlled by the salivary clock identified using chromatin immunoprecipitation (ChIP) assays, 22 of them showed reasonable transcriptional responses according to a DNA array in a salivary model system. Among these 22 genes, ARRB1, which is expressed in human salivary glands, was also expressed in model HSG cells at the transcriptional and translational levels. The profile of ARRB1 expression in human saliva was circadian, suggesting that ARRB1 could serve as a candidate circadian biomarker in saliva. We compared ARRB1 with other biomarkers in salivary samples from jet-lagged individuals. The circadian profile of ARRB1 reflected the time lag more than the profile of melatonin, whereas the profiles of cortisol and α-amylase did not reflect the time lag. Overall, these findings suggest that salivary ARRB1 could serve as a candidate biomarker that could be used to monitor the internal body clock.

Conflict of interest statement

The authors declare no competing interests.

Figures

Fig. 1
Fig. 1
Circadian clock in human salivary gland cells. a Circadian transcription of the BMAL1 gene in HSG and HeLa cells. HSG and HeLa cells were transfected with the BMAL1 promoter construct (−201 to +24), stimulated with dexamethasone and analyzed using a Kronos AB-2500. The fitted curve data of the detrended results are representative of at least three independent experiments. b. Less RORα was expressed in HSG cells and human salivary gland tissues. Transcripts from HSG and HeLa cells stimulated with 100 nmol·L–1 dexamethasone and human salivary gland tissues were analyzed by RT-PCR
Fig. 2
Fig. 2
Selection of candidate circadian markers. a Flow chart of the selection process. b Evaluation of the ChIP-on-chip results for the NR1D1 region. Transfected HSG cells tagged with BMAL1 and REV-ERBα were harvested at 12 and 24 h after dexamethasone stimulation, and then ChIP was performed using anti-tagged antibodies. Mock-transfected cells without dexamethasone stimulation were processed in the same manner and are indicated as “–”. Precipitated DNA was analyzed using a NimbleGen Human ChIP–chip array, and the NR1D1 locus and the tiling region are indicated on the map. The red and yellow boxes in the map indicate regions that showed false discovery rates (FDR) of ≤ 0.05 and 0.1 < FDR ≤ 0.2, respectively. c. Venn diagram of the gene set intersect between BMAL1-bound cells at 12 h and REV-ERBα-bound cells at 24 h after dexamethasone stimulation in HSG cells. d Total RNA was prepared from HSG cells harvested at 12 h and 24 h after dexamethasone simulation, and expression was compared using an Agilent microarray. We found 1 394 and 1 197 genes that were up- and downregulated, respectively, at 12 h compared with their expression 24 h after stimulation. e Overlap between genes that might be controlled by the Clock system and genes that were significantly upregulated at 12 h compared to 24 h after stimulation
Fig. 3
Fig. 3
Selection of candidate clones. a Expression of clones from the human salivary gland. Transcripts from human salivary glands analyzed by RT-PCR. b Rhythmic transcription. Transcripts from HSG cells stimulated with 100 nmol·L–1 dexamethasone and analyzed by qRT-PCR
Fig. 4
Fig. 4
Circadian expression of ARRB1 protein. Protein expression was analyzed by western blotting in HSG cells after stimulation with 100 nmol·L−1 dexamethasone. ARRB1, with anti-ARRB1 antibody; ACT, with anti-ACTIN antibody. Values for ARRB1 protein were normalized to those of actin protein, and the peak value was set to 1. Values represent the mean ± SEM of triplicate assays
Fig. 5
Fig. 5
Expression of ARRB1 in normal human salivary glands. ARRB1 was expressed in serous acini, intercalated ducts, striated ducts and excretory ducts in the epithelium of normal parotid glands and submandibular glands. Immunoreactivity to ARRB1 in secreted material. Arrowheads indicate secretions in ducts. Upper panel, acini, intercalated ducts, and striated ducts; lower panel, excretory ducts in the parotid gland. a, c Haematoxylin and eosin staining; b, d, ARRB1. Original magnification × 400
Fig. 6
Fig. 6
Circadian expression of ARRB1 protein in human saliva. Protein expression in human saliva was analyzed by Western blotting. The amounts of ARRB1 protein were normalized to those of actin protein (ACT), and the peak value was set to 1. Each sample was collected at the indicated time point (JST). The line indicates the fitted cosine curve for the circadian rhythm. ac, male adults; df, female adults
Fig. 7
Fig. 7
Circadian rhythm in human saliva. Expression of ARRB1 a and AMY1A b proteins in human saliva analyzed by western blotting. The amounts of ARRB1 and AMY1A proteins were normalized to those of actin protein. The contents of cortisol c and melatonin d were determined. The peak value was set to 1, and the values are represented as the mean ± SEM of triplicate assays. The red and blue dots represent the samples obtained before and after travel to the region that was 3.5 h behind JST, respectively, (red and blue dots, normal and jet lag samples, respectively). Each sample was collected at the indicated time point (JST), and we denoted “Day 1” as the day on which sampling began both before and after jet lag

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