Transcriptional and Post-Transcriptional Regulation of Oxytocin and Vasopressin Gene Expression by CREB3L1 and CAPRIN2

Abstract

Introduction:Water homeostasis is achieved by secretion of peptide hormones arginine vasopressin (AVP) and oxytocin (OXT) that are synthesised by separate populations of magnocellular neurones (MCNs) in the hypothalamus. To further understand the molecular mechanisms that facilitate biosynthesis of AVP and OXT by MCNs, we have explored the spatiotemporal dynamic, of two genes identified by our group as being important components of the osmotic defence response: Caprin2 and Creb3l1. Methods:By RNA in situ hybridization and immunohistochemistry, we have characterised the expression of Caprin2 and Creb3l1 in MCNs in the basal state, in response to dehydration, and during rehydration in the rat. Results:We found that Caprin2 and Creb3l1 are expressed in AVP and OXT MCNs and in response to dehydration expression increases in both MCN populations. Protein levels mirror the increase in transcript levels for both CREB3L1 and CAPRIN2. In view of increased CREB3L1 and CAPRIN2 expression in OXT neurones by dehydration, we explored OXT specific functions for these genes. By luciferase assays, we demonstrate that CREB3L1 may be a transcription factor regulating Oxt gene expression. By RNA immunoprecipitation assays and northern blot analysis of Oxt mRNA poly(A) tails, we have found that CAPRIN2 binds Oxt mRNA and regulates its poly(A) tail length. Moreover, in response to dehydration, Caprin2 mRNA is subjected to nuclear retention, possibly to regulate Caprin2 availability to the cytoplasm. Conclusion:The exploration of the spatiotemporal dynamics of Creb3l1 and Caprin2 encoded mRNAs and proteins has provided novel insights beyond the AVP-ergic system revealing novel OXT-ergic system roles of these genes in the osmotic defence response.

Transcription factor Creb3l1 maintains proteostasis in neuroendocrine cells

“…We have previously reported that Creb3l1 regulates the synthesis of Avp and Oxt in the SON [ [17] , [18] , [19] , [20] , [21] ] and extend these findings here to include Cartpt and Pdyn . This global response has similarities with studies of Creb3l1 in other secretory cell-types where the synthesis of secretory proteins and cell secretory capacity are reported to be controlled by this TF [ 23 , 24 , 26 , 43 ].…”

“…We were the first to describe expression of ER stress sensor cAMP responsive element-binding protein 3 like 1 (Creb3l1) in MCNs of the hypothalamus [ 17 ]. We have gone on to characterise its mechanism of activation and functions in MCNs in the regulation of vasopressin (AVP) and oxytocin (OXT) synthesis in response to increased physiological demand [ [16] , [17] , [18] , [19] , [20] , [21] ]. Creb3l1 is a TF of the CREB/ATF family that, like ATF6, is activated by regulated intramembrane proteolysis (RIP) [ 22 ].…”

Hypothalamic extraretinal photoreceptor Opsin3 regulates water balance, body temperature and motor activity

“…4 control and 4 WD rats were used for RNAscope analysis and for immunofluorescence based on previous experience (Barez-Lopez et al, 2022). 5 control and 5 Opn3 knockdown SON samples were used for RNA-seq analysis based on studies on power analysis and sample size estimation for RNA-Seq differential expression (Ching et al, 2014).…”

“…Fresh-frozen brains were sliced into 16 µm coronal sections and directly mounted on Superfrost Plus slides (Thermo Fisher Scientific, Waltham, MA, USA) and stored -80 C°. RNAscope in situ hybridization was performed as described (Barez-Lopez et al, 2022) using the probes Rn-AVP-C2 (Advanced Cell Diagnostics, 401421-C2) targeting 20 – 525 of rat Avp gene, Rn_OPN3 (Advanced Cell Diagnostics, 578411) targeting 418 – 1702 of rat Opn3 gene, and Rn-OXT-C3 (Advanced Cell Diagnostics, 479631-C3) targeting 3 – 493 of rat Oxt gene.…”

“…Images from RNAscope in situ hybridization studies were acquired with a Leica SP5-II AOBS confocal laser scanning microscope attached to a Leica DMI 6000 inverted epifluorescence microscope using a 63x PL APO CS lens with a 3.4-zoom factor. Quantification of Opn3 RNA dots in the nucleus (DAPI labelling close to either AVP- or OXT-positive cytoplasm) or cytoplasm (AVP or OXT labelling) of AVP or OXT neurones was performed using a modular workflow plugin for Fiji created by Dr Stephen J Cross from the Wolfson Bioimaging Facility of the University of Bristol, as described (Barez-Lopez et al, 2022). Images from immunofluorescence studies were acquired with a Leica SP8 AOBS confocal laser scanning microscope attached to a Leica DM I8 inverted epifluorescence microscope using a 40x HC PL APO CS2 lens.…”