Functional and morphological analysis of a novel peptide relaxin 3 in the central nervous system

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We have been investigating physiological function of a newly identified brain peptide at 2002 called, relaxin3 or insulin like peptide 7 (INSL7) which belongs to the insulin super family. Already we reported that relaxin 3 is dominantly expressed in neurons of the brainstem called nucleus incertus (NI) in the median dorsal pons (Tanaka M, et. al. Eur J Neurosci. 2005).1. First year, we studied the developmental and aged expression of relaxin 3 in the NI. Relaxin 3 mRNA was detected from embryonic day 18 by RT-PCR and in situ hybridization histochemistry. It was found at peptide level from birth. In aging, the number of relaxin 3 expressing neurons and level of expression at 18 month were decreased compared with those at 2 month. We also investigated the influence of serotonin (5-HT) because NI was innervated by 5-HT fibers and dorsal raphe nucleus, which contains abundant 5-HT neurons, is located just dorsal to the NI. Relaxin 3 neurons in the NI coexpressed 5-HT1A receptor and the tre … More atment of 5-HT depletor PCPA increased the relaxin 3 mRNA level. These results suggest 5-HT negatively regulate the relaxin 3 expression in the NI. The outcome of this study was published in the journal (Miyamoto et al, Regul Pept, 2008).2. Second year, as relaxin 3 mRNA was increased in various conditions such as stress and PCPA treatment, we started to study the mechanism of relaxin 3 transcription. We found that relaxin 3 was expressed in a mouse neuroblastoma cell line, Neuro2a, and investigated the intracellular signaling that activated relaxin 3 gene transcription in vitro. Using a clone stably transfected with a relaxin 3 promoter-EGFP gene, we observed that dibutyryl cyclic AMP and forskolin increased the relaxin 3 promoter activity. These increases were inhibited by pretreatment with a specific PKA inhibitor, H89. Moreover, the promoter activity was enhanced by CRF treatment after expression of CRF-Rl receptor on the cells. Taken together, these results indicate that relaxin 3 transcription is activated via the cAMP-PKA pathway in the downstream of CRF-Rl. The above results are now submitted to J Neurosci Res. Less