Assessment of the Role of Activator Protein-1 on Transcription of the Mouse Steroidogenic Acute Regulatory Protein Gene

Abstract

cAMP-dependent mechanisms regulate the steroidogenic acute regulatory (StAR) protein even though its promoter lacks a consensus cAMP response-element (CRE, TGACGTCA). Transcriptional regulation of the StAR gene has been demonstrated to involve combinations of DNA sequences that provide recognition motifs for sequence-specific transcription factors. We recently identified and characterized three canonical 5'-CRE half-sites within the cAMP-responsive region (-151/-1 bp) of the mouse StAR gene. Among these CRE elements, the CRE2 half-site is analogous (TGACTGA) to an activator protein-1 (AP-1) sequence [TGA(C/G)TCA]; therefore, the role of the AP-1 transcription factor was explored in StAR gene transcription. Mutation in the AP-1 element demonstrated an approximately 50% decrease in StAR reporter activity. Using EMSA, oligonucleotide probes containing an AP-1 binding site were found to specifically bind to nuclear proteins obtained from mouse MA-10 Leydig and Y-1 adrenocortical tumor cells. The integrity of the sequence-specific AP-1 element in StAR gene transcription was assessed using the AP-1 family members, Fos (c-Fos, Fra-1, Fra-2, and Fos B) and Jun (c-Jun, Jun B, and Jun D), which demonstrated the involvement of Fos and Jun in StAR gene transcription to varying degrees. Disruption of the AP-1 binding site reversed the transcriptional responses seen with Fos and Jun. EMSA studies utilizing antibodies specific to Fos and Jun demonstrated the involvement of several AP-1 family proteins. Functional assessment of Fos and Jun was further demonstrated by transfecting antisense c-Fos, Fra-1, and dominant negative forms of Fos (A-Fos) and c-Jun (TAM-67) into MA-10 cells, which significantly (P < 0.01) repressed transcription of the StAR gene. Mutation of the AP-1 site in combination with mutations in other cis-elements resulted in a further decrease of StAR promoter activity, demonstrating a functional cooperation between these factors. Mammalian two-hybrid assays revealed high-affinity protein-protein interactions between c-Fos and c-Jun with steroidogenic factor 1, GATA-4, and CCAAT/enhancer binding protein-beta. These findings demonstrate that Fos and Jun can bind to the TGACTGA element in the StAR promoter and provide novel insights into the mechanisms regulating StAR gene transcription.

Impact of Aging on Steroid Hormone Biosynthesis and Secretion

“…Under normal stress conditions the expression of major AP-1 constituent proteins [382,402] is up-regulated, but in the aging adrenal, the only transcription factor whose expression is increased (rather than decreased) is Jun B [387], a putative repressor of AP-1 function [406][407][408][409][410]. These events suggest that cellular defense against oxidative stress is reduced in aging animals, and the potential impact of this on steroidogenesis becomes even more clear when one realizes that promoter regions of StarD1/StAR and PBR/TSPO genes, the two intracellular molecules which assist in mediating the cholesterol transport process [53,58,59,66,76,81,[417][418][419], contain an AP-1 response element [420,421], and that the proximal AP-1 site in the StarD1/StAR promoter plays a pivotal role in regulating StarD1/StAR gene transcription [420].…”

Oxidative stress-induced inhibition of adrenal steroidogenesis requires participation of p38 mitogen-activated protein kinase signaling pathway

“…Transcriptional regulations of StAR have been extensively studied, and a cluster of transcription factors is involved in the regulatory process (Manna et al 2003, 2004, Hiroi et al 2004, Clem & Clark 2006, Manna & Stocco 2007. One of these transcription factors is AP-1, a dimeric complex composed primarily of the Fos and Jun family of proteins (Chinenov & Kerppola 2001, Hess et al 2004, which play a critical role in the regulation of StAR gene transcription (Manna et al 2004). Since CRE2 site overlaps with AP-1 site in the proximal StAR promoter region, its activity is also subject to regulation via the crosstalk between CREB and Fos/Jun proteins (Manna et al 2004, Manna & Stocco 2007.…”

“…One of these transcription factors is AP-1, a dimeric complex composed primarily of the Fos and Jun family of proteins (Chinenov & Kerppola 2001, Hess et al 2004, which play a critical role in the regulation of StAR gene transcription (Manna et al 2004). Since CRE2 site overlaps with AP-1 site in the proximal StAR promoter region, its activity is also subject to regulation via the crosstalk between CREB and Fos/Jun proteins (Manna et al 2004, Manna & Stocco 2007. Interestingly, studies on the transcriptional regulation of another cholesterol transport protein, PBR/TSPO (which is believed to work in concert with StAR (Miller 2007), have also identified an AP-1 binding site in its proximal promoter region (Giatzakis et al 2007).…”

Jun proteins modulate the ovary-specific promoter of aromatase gene in ovarian granulosa cells via a cAMP-responsive element

“…In fact, JunB actually moderately stimulated the synthetic AP1-responsive promoter and the collagenase promoter (compare column 1 with 2, and 3 with 4 in Figure 3a), suggesting that the Jun-mediated repression was promoter-specific. It was recently reported that transcription of the mouse steroidogenic acute regulatory protein (StAR) gene, another important gene involved in ovarian steroidogenesis, was stimulated by AP1 proteins in mouse Leydig cells (Manna et al, 2004). When tested in KGN cells, basal transcription from the same promoter was also stimulated by JunB and c-Jun (compare column 1 with 3 and 5 in bottom panel of Figure 3b).…”