Cyclic Adenosine 3′,5′-Monophosphate (cAMP) Enhances cAMP-Responsive Element Binding (CREB) Protein Phosphorylation and Phospho-CREB Interaction with the Mouse Steroidogenic Acute Regulatory Protein Gene Promoter

Abstract

Steroidogenic acute regulatory protein (StAR) transcription is regulated through cAMP-protein kinase A-dependent mechanisms that involve multiple transcription factors including the cAMP-responsive element binding protein (CREB) family members. Classically, binding of phosphorylated CREB to cis-acting cAMP-responsive elements (5'-TGACGTCA-3') within target gene promoters leads to recruitment of the coactivator CREB binding protein (CBP). Herein we examined the extent of CREB family member phosphorylation on protein-DNA interactions and CBP recruitment with the StAR promoter. Immunoblot analysis revealed that CREB, cAMP-responsive element modulator (CREM), and activating transcription factor (ATF)-1 are expressed in MA-10 mouse Leydig tumor cells, yet only CREB and ATF-1 are phosphorylated. (Bu)2cAMP treatment of MA-10 cells increased CREB phosphorylation approximately 2.3-fold within 30 min but did not change total nuclear CREB expression levels. Using DNA-affinity chromatography, we now show that CREB and ATF-1, but not CREM, interact with the StAR promoter, and this interaction is dependent on the activator protein-1 (AP-1) cis-acting element within the cAMP-responsive region. In addition, (Bu)2cAMP-treatment increased phosphorylated CREB (P-CREB) association with the StAR promoter but did not influence total CREB interaction. In vivo chromatin immunoprecipitation assays demonstrated CREB binding to the StAR proximal promoter is independent of (Bu)2cAMP-treatment, confirming our in vitro analysis. However, (Bu)2cAMP-treatment increased P-CREB and CBP interaction with the StAR promoter, demonstrating for the first time the physical role of P-CREB:DNA interactions in CBP recruitment to the StAR proximal promoter.

cAMP-independent signaling regulates steroidogenesis in mouse Leydig cells in the absence of StAR phosphorylation

“…As illustrated in Fig. 9A, mLTC-1 cells treated with different factors for 30 min (Clem et al 2005, Manna et al 2006, increased P-CREB to varying levels. P-CREB appeared as a doublet and was enhanced approximately 2$2-, 3$1-, 1$9-, 2$7-, 1$8-, 2$9-and 4$8-fold over controls in response to IGF-I, EGF, FGF, TGFa, IL-1, CSF-1, and hCG respectively.…”

“…These studies demonstrated that the protein kinase C (PKC) signaling plays an important role in regulating StAR expression and steroidogenesis (Pilon et al 2003, as has the mitogen-activated protein kinase (MAPK) family of Ser-Thr kinases (Gyles et al 2001, Seger et al 2001, Manna et al 2002a, Martinelle et al 2004, Martinat et al 2005. In addition, StAR's transcriptional regulation has been shown to be mediated by several transcription factors, including the cAMP-response element-binding protein (CREB) and dosage-sensitive sex reversal, adrenal hypoplasia congenita, critical region on the X chromosome, gene 1 (DAX-1) (Manna et al 2002b, 2006, Clem et al 2005. In addition, the scavenger receptor class B type 1 (SR-B1), a high-density lipoprotein receptor, was shown to be involved in regulating steroidogenesis by facilitating the mobilization of precursor cholesterol into steroidogenic cells (Rao et al 2003, Manna et al 2006.…”

Phosphoenolpyruvate Carboxykinase and Glucose-6-phosphatase Are Required for Steroidogenesis in Testicular Leydig Cells

“…MA-10 cells were kindly provided by Dr. Mario Ascoli and maintained as described previously (33,34). These well studied mouse Leydig tumor cells produce progesterone as the major steroid in response to both LH and cAMP analogs.…”

The ATF/CREB Family of Transcription Factors in Breast Cancer

“…Besides its effects on the immune system, ATF1 may also play a role in breast cancer via its regulation of steroidal hormone synthesis. Clem et al (Clem, Hudson, Clark 2005) showed that ATF1 binds the steroidal acute regulatory protein (StAR) promoter, although they failed to determine whether this association activates or blocks transcription. Regardless of ATF1's effect, regulation of StAR, an enzyme necessary for estradiol synthesis, may play an important role in preventing breast cancer because a longer time of exposure to estradiol (menarche to menopause) is correlated with increased breast cancer risk.…”

“…Houvras et al (Houvras and others 2000) found that wild type (wt) BRCA1 bound ATF1, causing transcription of a luciferase reporter gene. Such a finding strongly suggests (Ishiguro, 1997) StAR (Clem, 2005) Thrombospondin 1 (Ghoneim, 2007) Collagen, type X, α1 (Tsuchimochi, 2010) MMP13 (Tsuchimochi, 2010) Cyclin A (van Dam, 2001) Aromatase (Deb, 2006) MMP2 (Song, 2006) COX2 (Subbaramaiah, 2002) Cyclin D1 (Lee, 1999) Maspin (Maekawa, 2008) IFNγ (Xue, 2005) FoxP3 Fibronectin (Yin, 2010) Snail (Yin, 2010) Twist (Yin, 2010) Slug (Yin, 2010) av integrin (Yin, 2010) β6 integrin (Yin, 2010) E-cadherin (Yin, 2010) Increased malignancy Osteocalcin ( www.intechopen.com very few studies have been performed on the role of ATF1 in breast cancer. Important future studies will elucidate the effects of ATF1 on immunity and hormone synthesis in both normal and neoplastic breast cells, as well as its role in breast cancer in vivo.…”