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The cAMP-responsive element binding protein (CREB) regulates the expression of acid ceramidase (ASAH1) in H295R human adrenocortical cells.

ABSTRACT: Acid ceramidase (encoded by ASAH1) is a lipid hydrolase that catalyzes the conversion of ceramide (cer) into sphingosine (SPH) and a free fatty acid. Adrenocortical steroidogenesis is regulated by the trophic peptide hormone adrenocorticotropin (ACTH), which induces the expression of steroidogenic genes in the human adrenal cortex primarily via a cAMP/protein kinase A (PKA)-dependent pathway. ACTH also stimulates sphingolipid metabolism in H295R adrenocortical cells leading to changes in steroidogenic gene expression. Based on our previous data identifying SPH as an antagonist for the nuclear receptor steroidogenic factor 1 (SF-1) and the role of ACTH-stimulated changes in sphingolipid metabolism on steroidogenic gene transcription, the aim of the current study was to determine the role of ACTH signaling in regulating the expression of the ASAH1 gene in H295R cells. We show that activation of the ACTH signaling pathway induces ASAH1 gene expression by stimulating the binding of the cAMP-responsive element binding protein (CREB) to multiple regions of the ASAH1 promoter. CREB binding promotes the recruitment of the coactivators CREB binding protein (CBP) and p300 to the CREB-responsive regions of the promoter. Consistent with transcriptional activation, we show that cAMP signaling increases the trimethylation of Lys 4 on histone H3 (H3K4) along the ASAH1 promoter. Finally, RNA interference (RNAi) experiments demonstrate that CREB is indispensable for cAMP-induced ASAH1 transcription. These data identify the ACTH/cAMP signaling pathway and CREB as transcriptional regulators of the ASAH1 gene in the human adrenal cortex.

SUBMITTER: Lucki N

PROVIDER: S-EPMC3976422 | biostudies-literature | 2009 Aug

REPOSITORIES: biostudies-literature

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The cAMP-responsive element binding protein (CREB) regulates the expression of acid ceramidase (ASAH1) in H295R human adrenocortical cells.

Lucki Natasha N Sewer Marion B MB

Biochimica et biophysica acta 20090316 8

Acid ceramidase (encoded by ASAH1) is a lipid hydrolase that catalyzes the conversion of ceramide (cer) into sphingosine (SPH) and a free fatty acid. Adrenocortical steroidogenesis is regulated by the trophic peptide hormone adrenocorticotropin (ACTH), which induces the expression of steroidogenic genes in the human adrenal cortex primarily via a cAMP/protein kinase A (PKA)-dependent pathway. ACTH also stimulates sphingolipid metabolism in H295R adrenocortical cells leading to changes in steroid ...[more]

PMID: 19298866

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Project description:The mechanism of action of retinoic acid (RA) is of broad relevance to cell and developmental biology, nutrition, and cancer chemotherapy. RA is known to induce expression of the Burkitt's lymphoma receptor 1 (BLR1) gene which propels RA-induced cell cycle arrest and differentiation of HL-60 human myeloblastic leukemia cells, motivating the present analysis of transcriptional regulation of blr1 expression by RA. The RA-treated HL-60 cells used here expressed all RA receptor (RAR) and retinoid X receptor (RXR) subtypes (as detected by Northern analysis) except RXRgamma. Treatment with RAR- and RXR-selective ligands showed that RARalpha synergized with RXRalpha to transcriptionally activate blr1 expression. A 5'-flanking region capable of supporting RA-induced blr1 activation in HL-60 cells was found to contain a 205-bp sequence in the distal portion that was necessary for transcriptional activation by RA. Within this sequence DNase I footprinting revealed that RA induced binding of a nuclear protein complex to an element containing two GT boxes. Electromobility shift assays (EMSAs) and supershift assays showed that this element bound recombinant RARalpha and RXRalpha. Without RA there was neither complex binding nor transcriptional activation. Both GT boxes were needed for binding the complex, and mutation of either GT box caused the loss of transcriptional activation by RA. The ability of this cis-acting RAR-RXR binding element to activate transcription in response to RA also depended on downstream sequences where an octamer transcription factor 1 (Oct1) site and a nuclear factor of activated T cells (NFATc) site between this element and the transcriptional start, as well as a cyclic AMP response element binding factor (CREB) site between the transcriptional start and first exon of the blr1 gene, were necessary. Each of these sites bound its corresponding transcription factor. A transcription factor-transcription factor binding array analysis of nuclear lysate from RA-treated cells indicated several prominent RARalpha binding partners; among these, Oct1, NFATc3, and CREB2 were identified by competition EMSA and supershift and chromatin immunoprecipitation assays as components of the complex. RA upregulated expression of these three factors. In sum the results of the present study indicate that RA-induced expression of blr1 expression depends on a novel RA response element. This cis-acting element approximately 1 kb upstream of the transcriptional start consists of two GT boxes that bind RAR and RXR in a nuclear protein complex that also contains Oct1, NFATc3, and CREB2 bound to their cognate downstream consensus binding sites.

Dataset Information

The cAMP-responsive element binding protein (CREB) regulates the expression of acid ceramidase (ASAH1) in H295R human adrenocortical cells.

Publications

The cAMP-responsive element binding protein (CREB) regulates the expression of acid ceramidase (ASAH1) in H295R human adrenocortical cells.

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