Peripheral clock system circadian abnormalities in Cushing’s disease

ABSTRACT

In Cushing’s syndrome, the cortisol rhythm is impaired and can be associated with the disruption in the rhythmic expression of clock genes. In this study, we evaluated the expression of CLOCK, BMAL1, CRY1, CRY2, PER1, PER2, PER3 genes in peripheral blood leukocytes of healthy individuals (n = 13) and Cushing’s disease (CD) patients (n = 12). Participants underwent salivary cortisol measurement at 0900 h and 2300 h. Peripheral blood samples were obtained at 0900 h, 1300 h, 1700 h, and 2300 h for assessing clock gene expression by qPCR. Gene expression circadian variations were evaluated by the Cosinor method. In healthy controls, a circadian variation in the expression of CLOCK, BMAL1, CRY1, PER2, and PER3 was observed, whereas the expression of PER1 and CRY2 followed no specific pattern. The expression of PER2 and PER3 in healthy leukocytes presented a late afternoon acrophase, similarly to CLOCK, whereas CRY1 showed night acrophase, similarly to BMAL1. In CD patients, the circadian variation in the expression of clock genes was lost, along with the abolition of cortisol circadian rhythm. However, CRY2 exhibited a circadian variation with acrophase during the dark phase in patients. In conclusion, our data suggest that Cushing’s disease, which is characterized by hypercortisolism, is associated with abnormalities in the circadian pattern of clock genes. Higher expression of CRY2 at night outlines its putative role in the cortisol circadian rhythm disruption.     

A Molecular Clock Regulates Angiopoietin-Like Protein 2 Expression

Various physiological and behavioral processes exhibit circadian rhythmicity. These rhythms are usually maintained by negative feedback loops of core clock genes, namely, CLOCK, BMAL, PER, and CRY. Recently, dysfunction in the circadian clock has been recognized as an important foundation for the pathophysiology of lifestyle-related diseases, such as obesity, cardiovascular disease, and some cancers. We have reported that angiopoietin-like protein 2 (ANGPTL2) contributes to the pathogenesis of these lifestyle-related diseases by inducing chronic inflammation. However, molecular mechanisms underlying regulation of ANGPTL2 expression are poorly understood. Here, we assess circadian rhythmicity of ANGPTL2 expression in various mouse tissues. We observed that ANGPTL2 rhythmicity was similar to that of the PER2 gene, which is regulated by the CLOCK/BMAL1 complex. Promoter activity of the human ANGPTL2 gene was significantly induced by CLOCK and BMAL1, an induction markedly attenuated by CRY co-expression. We also identified functional E-boxes in the ANGPTL2 promoter and observed occupancy of these sites by endogenous CLOCK in human osteosarcoma cells. Furthermore, Cry-deficient mice exhibited arrhythmic Angptl2 expression. Taken together, these data suggest that periodic expression of ANGPTL2 is regulated by a molecular clock.     

Circadian gene methylation in rotating-shift nurses: a cross-sectional study

Investigating the methylation status of the circadian genes may contribute to a better understanding of the shift work-related circadian disruption in individuals exposed to artificial light at night. In the present study, we determined the methylation status of the circadian genes associated with a shift work pattern among nurses and midwives participating in a cross-sectional study in Lodz, Poland.

Quantitative methylation polymerase chain reaction assays were used to assess promoter CpG methylation in PER1, PER2, PER3, CRY1, CRY2, BMAL1, CLOCK, and NPAS2 in genomic DNA from whole blood of 347 women having a rotating-shift work schedule and 363 women working days only. The percentage of methylated reference (PMR) was assessed using fluorescent probes for PER1, PER2, PER3, CRY1, and NPAS2, and the percentage of gene methylation, as the methylation index (MI), using two sets of primers for BMAL1, CLOCK, and CRY2.

We tested the possible association between current and lifetime rotating night-shift work characteristics and circadian gene methylation by using proportional odds regression model with blood DNA methylation, categorized into tertiles, and adjusted for age, current smoking status, folate intake and blood collection time. The findings indicated that CpG methylation in PER2 promoter was significantly decreased (P < 0.004) among nurses and midwives currently working rotating shifts, as compared with day-working nurses and midwives. The lower percentage of PER2 methylation was associated with a higher monthly frequency of current night duties (2–7 night shifts, and eight or more night shifts per month) (P = 0.012) and was associated at borderline significance (P = 0.092) with the lifetime duration of shift work (>10 ≤ 20 years and >20 ≤ 43 years of rotating-shift work) among nurses and midwives (N = 710). Moreover, women with a longer lifetime duration of shift work presented a lower status of PER1 methylation (P = 0.040) than did the women with up to 10 years of rotating-shift work. Long lifetime duration of shift work (> 10 years) among current rotating night-shift workers (N = 347) was associated with BMAL1 hypomethylation (P = 0.013).

Among eight of the investigated circadian genes, only PER1, PER2, and BMAL1 showed differential methylation attributable to the rotating-shift work of nurses and midwives. The findings on blood-based DNA methylation in the circadian genes may provide a better insight into the mechanistic principles underlying the possible health effects of night-shift work but these should be verified in further studies recruiting larger populations of shift workers.