Identification of a Functional AP1 Element in the Rat Vasopressin Gene Promoter

Department of Endocrinology, Metabolism and Nephrology, Kochi University, Kôti, Kōchi, Japan
Endocrinology (Impact Factor: 4.5). 07/2006; 147(6):2850-63. DOI: 10.1210/en.2005-1222
Source: PubMed


Arginine vasopressin (AVP) is expressed in paraventricular, supraoptic, and suprachiasmatic nuclei of the hypothalamus, where transcription of the AVP gene is activated by various forms of stress, such as hyperosmolality, inflammation, and photic stimulation. In vasopressinergic neurons, the expression of the Fos/Jun family proteins is known to be rapidly induced after these stimuli as well. However, it is still unknown whether these proteins actually mediate AVP gene expression. In this study we examined in vitro the role of Fos/Jun protein in transcriptional regulation of the AVP gene using the BE(2)M17 neuroblastoma cell line. We found that 5'-promoter activity of the rat AVP gene (-803/+26) markedly increased when all combinations of the Fos/Jun family proteins were overexpressed. Coexpression of the cAMP-responsive element-binding protein-binding protein and steroid receptor coactivator-1a further enhanced the Fos/Jun-mediated transcription. Using site-directed mutagenesis and EMSA techniques, we identified an activation protein 1 (AP1)-like element (-134/-128; TGAATCA) in the AVP gene 5'-promoter region, which is the sole responsible site for the Fos/Jun-mediated transcription. We also found that 12-O-tetradecarbonyl phorbol 13-acetate stimulates AVP gene transcription partly via the AP1 site through the activation of ERK signaling. Together, these results suggest that a variety of Fos/Jun family member proteins stimulate transcription of the AVP gene through the AP1 site we identified. Furthermore, this effect may be activated by both protein kinase A and protein kinase C signaling pathways.

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    • "The biological significance of the greater expression of RORA and AP1 mRNAs in Oxt MCNs is unclear at present, but it is interesting that orphan nuclear hormone receptors such as RORA have been hypothesized to play a role in Oxt gene expression [51], [52]. However, while c-jun has been proposed to be a regulator of Avp gene expression [53], [54] but not Oxt gene expression, the greater expression of this TF in Oxt MCNs does not correlate with this association. Since TFs have many targets in a MCN other than the Oxt or Avp genes, and the expression of any given gene is likely to be determined by more than one TF, it is unlikely that the specific TF’s expression in the cell will reflect only the expression of the specific neuropeptide gene in the cell. "
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    ABSTRACT: The oxytocin (Oxt) and vasopressin (Avp) magnocellular neurons (MCNs) in the hypothalamus are the only neuronal phenotypes that are present in the supraoptic nucleus (SON), and are characterized by their robust and selective expression of either the Oxt or Avp genes. In this paper, we take advantage of the differential expression of these neuropeptide genes to identify and isolate these two individual phenotypes from the rat SON by laser capture microdissection (LCM), and to analyze the differential expression of several of their transcription factor mRNAs by qRT-PCR. We identify these neuronal phenotypes by stereotaxically injecting recombinant Adeno-Associated Viral (rAAV) vectors which contain cell-type specific Oxt or Avp promoters that drive expression of EGFP selectively in either the Oxt or Avp MCNs into the SON. The fluorescent MCNs are then dissected by LCM using a novel Cap Road Map protocol described in this paper, and the purified MCNs are extracted for their RNAs. qRT-PCR of these RNAs show that some transcription factors (RORA and c-jun) are differentially expressed in the Oxt and Avp MCNs.
    Full-text · Article · Jul 2013 · PLoS ONE
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    • "Many experiments to validate these TF and TFBS predictions were performed in heterologous tissue culture models in vitro and showed that many of these were potentially functional regulatory motifs in the Avp gene promoter [2]. These include one or more AP2 sites between −84 bp to 146 bp [45], AP1 sites at between −134 to −128 bp [48], [49], CRE between −227 and −116 bp [45], [50], [51], E box between −155 to −1341 bp [48], [52], [53], and SP 1 between −195 to −68 bp [48]. Interestingly, all of these which were predicted and in many cases functionally validated in vitro in the Avp promoter were located within the −288 to −116 bp domain that we propose contain the cell-type specific regulatory elements in the Avp gene (Fig. 9). "
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    ABSTRACT: The magnocellular neurons (MCNs) in the supraoptic nucleus (SON) of the hypothalamus selectively express either oxytocin (Oxt) or vasopressin (Avp) neuropeptide genes. In this paper we examine the cis-regulatory domains in the Avp gene promoter that are responsible for its cell-type specific expression. AAV vectors that contain various Avp gene promoter deletion constructs using EGFP as the reporter were stereotaxically injected into the rat SON. Two weeks following the injection immunohistochemical assays of EGFP expression from these constructs were done to determine whether the expressed EGFP reporter co-localizes with either the Oxt- or Avp-immunoreactivity in the MCNs. The results identify three major enhancer domains located at -2.0 to -1.5 kbp, -1.5 to -950 bp, and -950 to -543 bp in the Avp gene promoter that regulate the expression in Avp MCNs. The results also show that cell-type specific expression in Avp MCNs is maintained in constructs containing at least 288 bp of the promoter region upstream of the transcription start site, but this specificity is lost at 116 bp and below. Based on these data, we hypothesize that the -288 bp to -116 bp domain contains an Avp MCN specific activator and a possible repressor that inhibits expression in Oxt-MCNs, thereby leading to the cell-type specific expression of the Avp gene only in the Avp-MCNs.
    Full-text · Article · Nov 2012 · PLoS ONE
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    • "could bind to the putative AP-1/CRE binding sites in the AVP promoter [9] [21] [35] and possibly affect the transcriptional activities [35]. However, it is still unclear whether AP-1 complex induced in the SON binds to the AVP promoter to play any physiological role in the regulation of AVP gene transcription. "
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    ABSTRACT: While it is well known that osmotic stimulation induces the expression of Fos family members in the supraoptic nucleus (SON), it is unclear whether the induced protein products are involved in the regulation of the gene transcription of arginine vasopressin (AVP). In the present study, we examined the in vivo correlation between changes in AVP gene transcription and expression of the various Fos family members in the SON after acute osmotic stimuli. The data demonstrated that the peak of AVP transcription (measured by intronic in situ hybridization) observed 15min after an injection of hypertonic saline preceded the expression of Fos proteins, which became detectable at 30min and peaked at 120min. Electrophoretic mobility shift assay showed that the expressed Fos proteins bound to the composite AP-1/CRE-like site in the AVP promoter. These data suggest that Fos proteins in the SON induced by acute osmotic stimuli could affect AVP gene transcription by binding to the AVP promoter, but they are not prerequisite for the induction of AVP gene transcription.
    Full-text · Article · Dec 2010 · Neuroscience Letters
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