Clock genes show circadian rhythms in salivary glands

Abstract

Circadian rhythms are endogenous self-sustained oscillations with 24-hour periods that regulate diverse physiological and metabolic processes through complex gene regulation by "clock" transcription factors. The oral cavity is bathed by saliva, and its amount and content are modified within regular daily intervals. The clock mechanisms that control salivary production remain unclear. Our objective was to evaluate the expression and periodicity of clock genes in salivary glands. Real-time quantitative RT-PCR, in situ hybridization, and immunohistochemistry were performed to show circadian mRNA and protein expression and localization of key clock genes (Bmal1, Clock, Per1, and Per2), ion and aqua channel genes (Ae2a, Car2, and Aqp5), and salivary gland markers. Clock gene mRNAs and clock proteins were found differentially expressed in the serous acini and duct cells of all major salivary glands. The expression levels of clock genes and Aqp5 showed regular oscillatory patterns under both light/dark and complete-dark conditions. Bmla1 overexpression resulted in increased Aqp5 expression levels. Analysis of our data suggests that salivary glands have a peripheral clock mechanism that functions both in normal light/dark conditions and in the absence of light. This finding may increase our understanding of the control mechanisms of salivary content and flow.

Figure 1.

Clock gene RNAs (Per1, Per2, Bmal1, and Clock) were detected in both mouse SGs and the kidney. The SG marker (Smgc) was expressed only in SGs. SGs, salivary glands; kid, kidney; NC, negative control.

Figure 2.

Clock gene products localization in SGs. Immunohistochemistry results showed that Bmal1 (A-C), Clock (D-F), Per1 (G-I), and Per2 (J-L) proteins were expressed in the nucleus of both acinar cells and in the epithelial cells of the ducts in mouse SGs. Expression was much stronger in duct cells. The same expression pattern was detected in human SGs (O). Bmal1 and Per1 RNAs were detected in the nuclear of serous acini and duct cells of mouse SGs by in situ hybridization (M, N). MA, mucous acini; SA, serous acini; SD, striated ducts; PSG, parotid salivary gland; SLSG, sublingual salivary gland; SMSG, submandibular salivary gland. Bars = 50 µm in A, B, D, E, G, H, J, K; = 20 µm in C, F, I, L, M-O.

Figure 3.

Analysis of real-time PCR data showed that Clock, Bmal1, Per1, and Per2 RNAs are expressed in a rhythmic circadian manner in SGs under light/dark and dark/dark conditions (A-H). The mRNA levels are expressed as means of SE (n = 3 mice per time-point). All time interval calculations are based at the indicated zeitgeber (ZT, an event that provides the settings for a biological clock), and 6:00 a.m. was considered ZT 0.

Figure 4.

Expression of potential downstream targets of clock genes. Analysis of real-time PCR data showed that Aqp5 RNA is expressed in a circadian manner in SGs under light/dark and dark/dark conditions (A, B). In contrast, Ae2a showed a circadian pattern only in light/dark conditions (C) but not in complete-dark conditions (D). The AQP5 mRNA level in HEK293 was up-regulated after Bmal1 overexpression. All time interval calculations were based at the indicated zeitgeber (ZT 0 was considered to be 2 hrs after cell-cycle synchronization). The experiments were repeated 3 times, and 1 representative experiment is presented (*p < 0.05; compared with 0 hr).