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Abstract
Corticotropin Releasing Factor (CRF) is a neuropeptide, synthesized primarily by the paraventricular nucleus of the hypothalamus in the mammalian brain. It is the primary chemical messenger by which the body controls the activity of the Pituitary-Adrenal axis and is therefore ultimately responsible for orchestrating an endocrine response against stress. In an attempt to understand the molecular biology of CRF transcription, two different approaches were used.
The first approach involved the use of a transgenic mouse model. A construct containing the NPT II (Neo) reporter gene hooked to 5kb of the proximal promoter sequences from the human CRF gene was used to generate four independent transgenic mouse lines. The expression pattern of the transgene was studied using RNase Protection. The transgene was expressed in the appropriate tissues. Furthermore, in three of the transgenic mouse lines, a stress dependent increase was observed in the expression of the hCRF-Neo reporter. The transgene also showed diurnal variations in expression levels similar to that observed with the endogenous CRF gene, and this diurnal rhythm was suppressed by dexamethasone treatment. A sexual dimorphism in expression levels was also observed in the transgenic lines, with the females consistently showing elevated levels of the transgene. However, evaluation of transgenic placentas, showed no expression of the hCRF transgene similar to the endogenous mouse CRF gene, rather than reflecting the human expression pattern.
The second approach involved the use of a human neuroblastoma cell line BE(2)-M17 which has been shown to express the endogenous CRF gene upon induction with retinoic acid. Retinoic acid treatment was shown to increase the expression of the POU protein Brn-2. This was confirmed both at the protein level and the mRNA level using, electromobility shift assays and RT-PCR respectively. To test for the requirement of Brn-2 for CRF expression, antisense expression constructs of Brn-2 were transfected into the cell line. An antisense Brn-2 dose dependent decrease in the induction of CRF expression was observed. However, using an expression vector, we observed that the presence of the Brn-2 protein was not sufficient for CRF expression and could not substitute for the retinoic acid treatment.
