Modulatory role and signaling pathways of cortisol on the temporal expression of clock genes in ZEM-2S cell line

• Grant number: 11/04048-2
• Support Opportunities: Scholarships in Brazil - Master
• Start date: February 01, 2012
• End date: January 31, 2014
• Field of knowledge: Biological Sciences - Physiology - Compared Physiology
• Principal Investigator: Ana Maria de Lauro Castrucci
• Grantee: Jennifer Caroline de Sousa
• Host Institution: Instituto de Biociências (IB). Universidade de São Paulo (USP). São Paulo , SP, Brazil
• Associated research grant: 06/03381-1 - Comparative physiology of peripheral clocks: clock genes (CLOCK, PER1, PER2, CRY1 and BMAL 1) and their modulation by light and hormones in fish, amphibians and mammals, AP.TEM

Abstract

Studies on the circadian expression of clock genes have suggested hormones as potential synchronizing agents. Regulation of rhythmicity of peripheral clocks is among the various physiological effects of glucocorticoids (GCs), which are candidate to zeitgeber, since their circulating levels present robust daily patterns and are one of the major outputs of the mammalian central pacemaker. However, in other vertebrates, such a feature has yet to be clarified as well as if the hormonal regulation is direct or indirect, what are its mechanisms and consequences. Applying qPCR technique, we evaluated the gene expression profile of per1b and cry1b, negative feedback loop members of the molecular machinery of biological clock, in ZEM-2S cells of the teleost Danio rerio. The cells were exposed to photoperiod regimen of 12:12 LD; kept in constant darkness (DD); kept in DD but subject to medium changes or treated with daily pulses of 10-7 M dexamethasone (10-7 M DEXA), a glucocorticoid synthetic analogue. In 12:12 LD, both genes presented temporal variation, with peaks of expression in the light phase. In DD, per1b showed an oscillatory profile with attenuated amplitude, whereas cry1b did not oscillate throughout 24 h. DEXA-free medium changes in constant DD conditions altered significantly per1b and cry1b expression profiles. Nevertheless, DEXA daily pulses promoted a temporal oscillation much more pronounced for per1b, modulating positively or negatively its expression at different time points, whereas cry1b was not susceptible to the hormone. RU 486 at 10-5 M abolished the peak of expression of per1b previously observed at ZT 16. On the other hand, RU 486 at 10-6 M increased the gene expression around 3-fold in comparison to just DEXA treatment. These results confirm ZEM-2S cells as a model of peripheral clocks in culture due to their intrinsec photosensitivity, emphasizing the light/dark cycle as a major synchronizing agent. The glucocorticoid modulates per1b expression, probably through a direct genomic pathway, without excluding however its possible action through membrane receptors, since RU 486 exerted agonistic activity. Glucocorticoids could, therefore, be one of the factors that regulate the complex molecular machinery of zebrafish biological clock. (AU)

Academic Publications

(References retrieved automatically from State of São Paulo Research Institutions)

SOUSA, Jennifer Caroline de. Glucocorticoid effects on clock gene expression in Danio rerio ZEM-2S cells. 2015. Master's Dissertation - Universidade de São Paulo (USP). Instituto de Biociências (IBIOC/SB) São Paulo.