Characterization of the molecular basis involved on the expression of circadian clock genes in the pituitary at different thyroid states and the consequences for the synthesis/secretion of anterior pituitary hormones

Abstract

The anterior pituitary gland occupies a central position in the hypothalamus-pituitary-gland/tissues axes, acting in the synthesis and secretion of hormones related to several biological functions as reproduction, growth and metabolism. The plasma concentration of pituitary hormones fluctuates over 24 h, but the molecular mechanism responsible for the generation and maintenance of these oscillations remains undefined. The generation/maintenance of circadian rhythmicity, central or peripheral, is supported by a set of genes called clock genes involved in an auto-regulatory transcription-(post)translation-based feedback loop in a period of 24 h. These genes are also involved in the regulation of several genes related to rhythmicity, hormonal response, growth regulation and cell death in anterior pituitary cells, regardless of the regulation mediated by the central clock located at the suprachiasmatic nuclei. It is known that in the hypothyroidism and hyperthyroidism, the GH, TSH and LH secretions are changed and, that the T3 availability in peripheral tissues is controlled by the activity of Deiodinase 2, which in turn, presents a circadian rhythm. Moreover, genomic actions of T3 are triggered after its binding to the thyroid hormone receptor that dimerizes with receptors belonging to the nuclear receptors superfamily, in which, the retinoic acid receptor (RXR) and the retinoic acid orphan-related receptor alpha (RORa) belong. The RORa is an important factor that modulates the Bmal1 expression, an important component of the clock. All these data suggest that the anterior pituitary cells present an intrinsic circadian clock and that the clock genes expression might be altered in different thyroidal states, direct or indirectly, changing the synthesis/secretion of anterior pituitary hormones. To investigate this hypothesis, we intend to: a) characterize the presence/functionality of the intrinsic circadian clock in the anterior pituitary; b) identify possible interactions between the circadian clock and the secretion of pituitary hormones; c) identify possible actions of thyroid hormones on the expression of clock genes in the anterior pituitary; d) characterize the molecular mechanisms involved in this possible interaction considering transcriptional and post-translational actions, as well as the involvement of microRNAs and; e) correlate the alterations observed in the different hypothalamic-pituitary-gland axes during the hypo- and hyperthyroidism with the circadian clock. We will use methods such as RT-qPCR, Bmal1 silencing with short hairpin RNA followed by RNAseq, in silico studies using bioinformatics tools to analyze the promoter regions, ChIP-PCR assay, and the content and degree of phosphorylation of the protein components of the clock models of rats submitted to hypothyroidism or hyperthyroidism in adulthood. (AU)