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. 2015 Oct 26;8(1):68.
doi: 10.1186/s13041-015-0159-1.

Transcription factor CREB3L1 mediates cAMP and glucocorticoid regulation of arginine vasopressin gene transcription in the rat hypothalamus

Mingkwan Greenwood  1 , Michael P Greenwood  2 , Andre S Mecawi  3   4   5 , Su Yi Loh  6 , José Antunes Rodrigues  7 , Julian F R Paton  8 , David Murphy  9   10
Affiliations

Transcription factor CREB3L1 mediates cAMP and glucocorticoid regulation of arginine vasopressin gene transcription in the rat hypothalamus

Mingkwan Greenwood et al. Mol Brain. .

Abstract

Background: Arginine vasopressin (AVP), a neuropeptide hormone that functions in the regulation of water homeostasis by controlling water re-absorption at kidneys, is synthesised in supraoptic nucleus and paraventricular nucleus of the hypothalamus. An increase in plasma osmolality stimulates secretion of AVP to blood circulation and induces AVP synthesis in these nuclei. Although studies on mechanism of AVP transcriptional regulation in hypothalamus proposed that cAMP and glucocorticoids positively and negatively regulate Avp expression, respectively, the molecular mechanisms have remained elusive. Recently, we identified CREB3L1 (cAMP-responsive element binding protein 3 like 1) as a putative transcription factor of Avp transcription in the rat hypothalamus. However the mechanism of how CREB3L1 is regulated in response of hyperosmotic stress in the neurons of hypothalamus has never been reported. This study aims to investigate effect of previously reported regulators (cAMP and glucocorticoid) of Avp transcription on transcription factor CREB3L1 in order to establish a molecular explanation for cAMP and glucocorticoids effect on AVP expression.

Results: The effect of cAMP and glucocorticoid treatment on Creb3l1 was investigated in both AtT20 cells and hypothalamic organotypic cultures. The expression of Creb3l1 was increased in both mRNA and protein level by treatment with forskolin, which raises intracellular cAMP levels. Activation of cAMP by forskolin also increased Avp promoter activity in AtT20 cells and this effect was blunted by shRNA mediated silencing of Creb3l1. The forskolin induced increase in Creb3l1 expression was diminished by combined treatment with dexamethasone, and, in vivo, intraperitoneal dexamethasone injection blunted the increase in Creb3l1 and Avp expression induced by hyperosmotic stress.

Conclusion: Here we shows that cAMP and glucocorticoid positively and negatively regulate Creb3l1 expression in the rat hypothalamus, respectively, and regulation of cAMP on AVP expression is mediated through CREB3L1. This data provides the connection between CREB3L1, a newly identified transcription factor of AVP expression, with the previously proposed mechanism of Avp transcription which extends our understanding in transcription regulation of Avp in the hypothalamus.

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Figures

Fig. 1
Fig. 1
cAMP and glucocorticoid regulate Creb3l1 expression. AtT20 cells were used to study regulation of Creb3l1. a–c AtT20 cells were treated with 10 μM FSK or vehicle (DMSO). a Creb3l1 mRNA expression was observed by qPCR at various time points (n = 3; One way ANOVA). b–c Immunoblotting was performed to examine protein expression level of CREB3L1 in both b total protein extracts and c cytosolic/nuclear extracts. d–e AtT20 cells were treated with vehicle (DMSO), 10 μM FSK and/or 100nM DEX at various time points. d Effect of glucocorticoid on c-Fos and Creb3l1 expression was examined by qPCR (n = 3; Two way ANOVA). e Immunoblotting of CREB3L1 was performed at 24 h after treatment. GAPDH was used as an internal control for immunoblotting. * in B and E indicates non-specific band in immunoblotting. * in D indicate significant from FSK + DEX compared to FSK group. Scale bar, 20 μm; Error bar, +SEM; *,p < 0.05, **, p < 0.01; ***, p < 0.001; FSK, forskolin; DEX, dexamethasone
Fig. 2
Fig. 2
Effect of cAMP activation and glucocorticoid in organotypic culture of rat hypothalamus. a Rat hypothalamic explants were cultured on Millicell membranes. An observation under bright field microscope showed the area of PVN and SCN are intact. b Immunofluorescent staining of AVP-NPII was performed at day 14 to demonstrate survival of vasopressinergic neurons in the culture. The high magnification image of the area in the box is shown. c The mRNA expression of hypothalamic explants in response to treatment of 10 μM FSK and/or 100nM DEX for 4 h (n = 5-7) and 24 h (n = 8-15) was examined by qPCR. Scale bar, 100 μm; Error bar + SEM; *, p < 0.05; **, p < 0.01; ***, p < 0.001 (One way ANOVA); FSK, forskolin; DEX, dexamethasone; PVN, paraventricular nucleus; SCN, suprachiasmatic nucleus. hnAvp, arginine vasopressin heteronuclear RNA
Fig. 3
Fig. 3
cAMP-activated AVP promoter activity is modulated through Creb3l1. a-b AtT20 cells were transfected with 1 kb rat Avp promoter construct. At 24 h after transfection the cells were a treated with 10 μM FSK for 4 and 24 h or b pre-treated with 100nM DEX or DMSO for 2 h and followed by 4 h treatment of 10 μM FSK or DMSO and luciferase activity was measured (n = 3). c–e The Creb3l1-knockdown AtT20 cell line was produced in parallel with a control non-targeting shRNA cell line by transduction of lentivirus containing Creb3l1 or control non-targeting shRNA and further selected with puromycin. c The level of Creb3l1 knockdown was investigated by qPCR and immunoblotting in the AtT20 shRNA cell lines (n = 3). d The effect of FSK on Creb3l1 mRNA expression was examined by qPCR in control non-targeting and Creb3l1 knockdown cell lines (n = 3). e Luciferase assays were performed in control and Creb3l1-knockdown cell lines using 1 kb Avp promoter construct driving luciferase reporter gene (n = 3-4). Error bar + SEM; *, p < 0.05; **, p < 0.01; ***, p < 0.001 (Two way ANOVA for A, D and E, One way ANOVA for B and independent sample unpaired Student’s t-tests for C). FSK, forskolin; Con, Control; sh, shRNA; h, hour; ns, no significant difference
Fig. 4
Fig. 4
Creb3l1 expression and localisation in rat PVN and SON in response to acute hyperosmotic stress. Rats were i.p. injected with hypertonic solution (1.5 M NaCl 1.5 ml/100 g body weight). a The level of c-Fos and Creb3l1 gene expression was examined by qPCR in rat PVN and SON (n = 5). b Immunofluorescent staining of CREB3L1 (green) and AVP (red) showed early activation of CREB3L1 protein in response of hypertonic injection. The high magnification images by confocal microscope show the change in localization of CREB3L1 from perinuclear area to cytoplasm and nucleus in hyperosmotic stress. Scale bar, 70 μm and 10 μm (for confocal). Error bar + SEM; **, p < 0.01; ***, p < 0.001 (One way ANOVA); PVN, paraventricular nucleus; SON, supraoptic nucleus; AVP, arginine vasopressin
Fig. 5
Fig. 5
Effect of dexamethasone on Creb3l1 expression in the rat PVN and SON. Rats were injected (i.p.) with either DEX or vehicle. Two hours after injection, isotonic (IS; 0.15 M NaCl) or hypertonic saline (HS; 1.5 M NaCl; 1.5 ml/100 g body weight) were administrated (i.p.). The brains were collected at 4 h after IS/HS injection. qPCR was performed to examine mRNA expression of Creb3l1 and Avp (n = 6-8). Error bar + SEM; *, p < 0.05; **, p < 0.01; ***, p < 0.001 (Two-way ANOVA), +, p < 0.05 (independent sample unpaired Student’s t-tests). DEX, dexamethasone; PVN, paraventricular nucleus; SON, supraoptic nucleus; hn, heteronuclear; Avp, arginine vasopressin
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References

    1. Burbach JP, Luckman SM, Murphy D, Gainer H. Gene regulation in the magnocellular hypothalamo-neurohypophysial system. Physiol Rev. 2001;81(3):1197–1267. - PubMed
    1. Bourque CW. Central mechanisms of osmosensation and systemic osmoregulation. Nat Rev Neurosci. 2008;9(7):519–531. doi: 10.1038/nrn2400. - DOI - PubMed
    1. McKinley MJ, McAllen RM, Davern P, Giles ME, Penschow J, Sunn N et al. The sensory circumventricular organs of the mammalian brain. Adv Anat Embryol Cell Biol. 2003;172:III-XII, 1–122, back cover. - PubMed
    1. Boone M, Deen PM. Physiology and pathophysiology of the vasopressin-regulated renal water reabsorption. Pflugers Arch. 2008;456(6):1005–1024. doi: 10.1007/s00424-008-0498-1. - DOI - PMC - PubMed
    1. Schmale H, Heinsohn S, Richter D. Structural organization of the rat gene for the arginine vasopressin-neurophysin precursor. EMBO J. 1983;2(5):763–767. - PMC - PubMed

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