Abstract
Cyclic AMP (cAMP) inducible transcription factor cAMP responsive element binding protein 3 like 1 (Creb3l1) is strongly activated in the hypothalamus in response to hyperosmotic cues such as dehydration (DH). We have recently shown that Creb3l1 expression is upregulated by cAMP pathways in vitro, however the exact mechanisms are not known. Here we show that increasing Creb3l1 transcription by raising cAMP levels in mouse pituitary AtT20 cells automatically initiates cleavage of Creb3l1, leading to a greater abundance of the transcriptionally active N-terminal portion. Inhibiting protein synthesis indicated that de novo protein synthesis of an intermediary transcription factor was required for Creb3l1 induction. Strategic mining of our microarray data from dehydrated rodent hypothalamus revealed four candidates, reduced to two by analysis of acute hyperosmotic-induced transcriptional activation profiles in the hypothalamus, and one, orphan nuclear receptor Nr4a1, by direct shRNA mediated silencing in AtT20 cells. We show that activation of Creb3l1 transcription by Nr4a1 involves interaction with a single NBRE site in the promoter region. The ability to activate Creb3l1 transcription by this pathway in vitro is dictated by the level of methylation of a CpG island within the proximal promoter/5′UTR of this gene. We thus identify a novel cAMP-Nr4a1-Creb3l1 transcriptional pathway in AtT20 cells and also, our evidence would suggest, in the hypothalamus.
Highlights
Transcription factor Cyclic AMP (cAMP) responsive element binding protein 3 like 1 (Creb3l1), known as old astrocyte -induced substance (OASIS), was first identified in long-term cultured astrocytes and gliotic tissue (Honma et al, 1999)
endoplasmic reticulum (ER) stress has been at the forefront of investigations surrounding regulation of Creb3l1 synthesis and cleavage by regulated intramembrane proteolysis (RIP) since this mechanism was first identified in C6 glioma cells (Murakami et al, 2006)
We show that Creb3l1 cleavage is constitutively active (CA) in AtT20 cells, with increased Creb3l1 transcription automatically leading to a greater abundance of N-terminal cleaved Creb3l1
Summary
Transcription factor cAMP responsive element binding protein 3 like 1 (Creb3l1), known as old astrocyte -induced substance (OASIS), was first identified in long-term cultured astrocytes and gliotic tissue (Honma et al, 1999). Creb3l1 is one of five members of the Creb basic leucine zipper domain transcription factor subfamily (Kondo et al, 2011). Transcriptional Regulation of Creb3l1 to liberate the N-terminal fragment (Murakami et al, 2006). This active form of Creb3l1 enters the nucleus to activate the transcription of target genes (Omori et al, 2002). Whilst it is well accepted that activation of Creb3l1, like other Creb family members, requires RIP (Chan et al, 2011), the mechanisms that mediate the physiological regulation of Creb3l1 are less well understood
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