February 2011 Newsletter:

Grant funding, Summer Student Project

A puzzle in retinoic acid regulation of ACTH in hypothalamus and pituitary.

Introduction:
Retinoic acid (RA) is a metabolite of retinol (vitamin A) and has important roles in embryonic development, cell development and survival. It binds and activates retinoic acid receptors (RARs), which belong to the family of nuclear receptors. The RA signalling pathway has recently been found in the hypothalamus1. Pro-opiomelanocortin (POMC) is a pro-hormone found in the hypothalamic regions of the brain that can be proteolysed to a range of neuropeptides. It is cleaved to adrenocortocotropic hormone (ACTH) by pro-hormone convertase 1 (PC1). ACTH can then be cleaved further by pro-hormone convertase 2 (PC2) to α-melanocyte-stimulating hormone (α-MSH)2. It has recently been shown that RA increases ACTH levels in the hypothalamus1, but can decrease ACTH in the pituitary3.The mechanism for these effects is unknown.

Aims:
The aim of the project was to investigate how RA regulates ACTH in the hypothalamus and pituitary.

Methods:
Three rats were injected with RA (10mg/kg/day) over a three day period, pituitary and hypothalamus tissue were collected from each animal and from three control animals which were not treated with RA. The same tissues were collected from rats kept on a long-day photoperiod (8h light, 16h dark in which RA signalling is high1) or a short-day photoperiod (16h dark, 8h light in which RA signalling is low1) these animals were perfused with 4% paraformaldehyde before tissue collection. Pituitaries were sectioned at 8-10 μm and hypothalami at 40 μm and immunohistochemistry was performed. RNA, DNA and protein were extracted from the hypothalamus and pituitary pieces using Qiagen AllPrep kit. Western blotting and qPCR were performed.

Results:
An anti-PC1 antibody was tested on hypothalamus and pituitary using immunohistochemistry and western blotting. qPCR was also performed to look at Pcsk1 gene. Immunohistochemistry and western blotting showed that the antibody did not work (results not shown). qPCR however was able to demonstrate that PC1 mRNA did not change significantly between control and RA treated (fig.1A and 1C).

Conclusion:
The levels of PC2 immunoreactivity were higher in the hypothalamus of long-day and RA treated animals, which could be because more RA is produced in the hypothalamus of rats kept on a long-day photoperiod. It also suggests that RA signalling may affect POMC regulation of ACTH and α-MSH.

References
1. Shearer, K.D., Goodman, T.H., Ross, A.W., Reilly, L., Morgan, P.J., McCaffery P.J. (2010). Photoperiodic regulation of retinoic acid signalling in the hypothalamus. Journal of Neurochemistry, 112, 246-257.

2. Pritchard, L.E., Turnbull, A.V., White, A. (2002). Pro-opiomelanocortin processing in the hypothalamus: impact on melanocortin signalling and obesity. Journal of Endocrinology, 172, 411-421.

3. Castillo V, Giacomini D, Páez-Pereda M, Stalla J, Labeur M, Theodoropoulou M, Holsboer F, Grossman AB, Stalla GK, Arzt E. (2006). Retinoic acid as a novel medical therapy for Cushing's disease in dogs. Endocrinology. 147, 4438-4444.

Aberdeen