Activation of the hypothalamo-pituitary-adrenal (HPA) axis during stress is associated with increased expression of genes that code for regulatory hormones such as corticotrophin-releasing factor (CRF) and ACTH. The identity of the transcription factors that mediate these changes in gene expression is not known. In the present study we have investigated the expression of the cAMP response-element binding protein (CREB) in mouse pituitary, and its regulation during a pharmacological paradigm that simulates activation of the CRF-ACTH axis. Using Western blots and DNA binding assays we have shown that both CREB protein (43 kDa) and CRE binding exhibit a readily-detectable basal level of activity in the pituitary. Following treatment with the 11 beta-hydroxylase inhibitor metyrapone, CRE binding activity was increased at 1 and 2 h but levels of CREB protein were not found to be consistently elevated. However, using a Ser133 phosphopeptide-specific antibody, that detects the functionally important phosphorylated form of CREB (P-CREB), we have shown that levels of pituitary P-CREB are markedly elevated following metyrapone. The same antibody was also used in DNA binding assays, and in the presence of this antiserum CRE binding activity in samples extracted from metyrapone-treated animals was reduced to levels similar to controls. Parallel experiments have confirmed previous studies showing increases in c-Fos expression and AP-1 DNA binding activity following metyrapone treatment but we have shown that c-Fos-associated binding activity does not appear to contribute to the increase in activity detected using the CRE binding probe. Our evidence of functionally relevant changes in pituitary CREB activity following glucocorticoid depletion must be viewed in the context of numerous other novel pituitary transcription factors that are implicated in HPA regulation, but our use of mice as an experimental model has facilitated the use of novel mouse mutants that can be used to dissect the role of individual factors.
PR Manna, DW Eubank, E Lalli, P Sassone-Corsi and DM Stocco
Transcriptional induction by cAMP is mediated through the interaction of the cAMP response-element binding protein (CREB) with a cAMP response element (CRE) in the promoter of target genes. The steroidogenic acute regulatory (StAR) protein gene is regulated by cAMP-mediated signaling in steroidogenic cells even though its promoter lacks a consensus CRE. Previously, we have identified three highly conserved 5'-CRE half-sites within the -96/-67 bp region of the mouse StAR gene, and a member of the CREB family (CREB/CRE modulator (CREM)) was shown to be involved in its expression and regulation. Here we show that CREB and CREMtau (but not CREMalpha and CREMbeta) have qualitatively similar effects on StAR promoter activity in response to (Bu)(2)cAMP. Studies on the effects of the functional integrity of the CRE half-sites on CREB-dependent (Bu)(2)cAMP-mediated StAR gene transcription demonstrated the greater importance of the CRE2 site in comparison with the CRE1 and CRE3 sites. The CRE2 sequence was also found to bind specifically to recombinant CREB protein and nuclear extract from MA-10 mouse Leydig tumor cells. The cAMP and CREB/CREM responsive region (-151/-1 bp) of the mouse StAR promoter also contains three recognition motifs for steroidogenic factor 1 (SF-1). Electrophoretic mobility shift assays and reporter gene analyses demonstrated the involvement of different SF-1 elements in StAR gene expression with the order of importance being SF-1/3>SF-1/1>SF-1/2. Specific mutations that eliminated the binding sites of CRE and SF-1 elements, either alone or in combination, resulted in an attenuation of StAR promoter activity, indicating that CREB and SF-1 can regulate StAR gene transcription in a cooperative fashion. In addition, mammalian two-hybrid assays revealed a high affinity protein-protein interaction between CREB/CREMtau and SF-1 which appeared to be dependent upon CREB protein phosphorylation. These findings further demonstrate CREB's role in StAR gene transcription and also provide evidence that the combined action of CREB/CREMtau and SF-1 results in enhanced activation of the StAR promoter.
M Fernández, F Sánchez-Franco, N Palacios, I Sánchez and L Cacicedo
In previous studies we demonstrated that vasoactive intestinal peptide (VIP) mediation, and interactions between mitogen-activated protein kinase (MAPK) and cAMP/protein kinase A (PKA) signaling pathways are implicated in insulin-like growth factor I (IGF-I)- and VIP-induced lactotroph proliferation. These facts led us to investigate the intracellular mechanisms involved in IGF-I- and VIP-induced lactotroph proliferation. Exposure of cultured male rat pituitary cells to IGF-I (10−7 M) or VIP (10−7 M) stimulated the MAPK cascade. Studies in GH4C1 cells, with an expression vector for Rap1 GTPase-activating protein (Rap1 GAP1), demonstrated reduced VIP-induced MAPK activation, indicating that VIP-dependent activation of the extracellular signal-regulated kinase (ERK) pathway requires PKA-Rap1 signaling. IGF-I induced cAMP-response element (CRE)-binding protein (CREB) phosphorylation through the Ras-MAPK pathway, whereas VIP phosphorylated CREB directly via PKA. The mechanisms that regulate IGF-I-and VIP-CREB-dependent gene transcription were examined using GH4C1 cells transiently transfected with a CRE reporter gene. IGF-I and VIP stimulation of CRE-mediated transcription required activation of both Ras-MAPK and cAMP/PKA signaling. This activation was blocked in the presence of Rap1 GAP1. In summary, we showed that IGF-I and VIP stimulated MAPK activity and the phosphorylation of CREB in pituitary cells. Furthermore, VIP-dependent activation of PKA-Rap1-ERK pathways mediated VIP and IGF-I effects on CREB-dependent transcription in GH4C1 cells. Thus, it is possible that VIP- and IGF-I-induced lactotroph proliferation may involve Rap1.
Feng Wang, Lu Wang, Yifeng Wang, Dai Li, Tianpeng Hu, Manyi Sun and Ping Lei
partly explain the effect of metabolic disorders on cognitive function in elderly ( Mukherjee et al . 2018 , Zhong et al . 2019 ). cAMP-response element-binding protein (CREB) is a transcriptional regulator in the nucleus of neurons, which plays an
Yuxiao Tang, Xingjian Cai, Hongwei Zhang, Hui Shen, Wanyin Wang, Zhilei Shen, Wei Gu, Changquan Ling and Min Li
mimics and inhibitors (Genepharma) at a concentration of 50 nM and 100 nM, respectively. We transfected plasmids for cAMP response element-binding protein (CREB; Genepharma) at a concentration of 3 μg/mL. We performed all transfections by using micropoly
Hseng-Kuang Hsu, Pei-Lin Shao, Ke-Li Tsai, Huei-Chuan Shih, Tzu-Ying Lee and Chin Hsu
sequence (5′-AGTTGAGGGGACTTTCCCAGGC-3′) or cAMP response element-binding protein (CREB) consensus binding sequence (5′-AGAGATTGCCTGAC GTCAGACAGCTAG-3′) was purchased from Promega and end-labeled with γ 32 P-ATP according to manufacturer’s recommendation
Young Sun Kang, Yun Gyu Park, Bo Kyung Kim, Sang Youb Han, Yi Hwa Jee, Kum Hyun Han, Mi Hwa Lee, Hye Kyoung Song, Dae Ryong Cha, Shin Wook Kang and Dae Suk Han
, MAP kinase kinase 3/6 (MKK3/6), phospho-specific MKK3/6, cAMP response element binding protein (CREB), and phospho-specific CREB (New England Biolabs, Inc., Beverly, MA, USA) diluted 1:1000 were applied to the membrane. The filter was then washed four
Amélie Gormand, Christine Berggreen, Lahouari Amar, Emma Henriksson, Ingrid Lund, Sebastian Albinsson and Olga Göransson
occurs partly through the cAMP/protein kinase A (PKA) pathway via the activation of cAMP-response element-binding protein (CREB). CREB is required for the differentiation of adipocytes through induction of the expression of C/EBPβ and potentially C
Pulak R Manna, Syam P Chandrala, Youngah Jo and Douglas M Stocco
, Manna et al. 2002 a , 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
Wei-An Lai, Yi-Ting Yeh, Wei-Ling Fang, Leang-Shin Wu, Nobuhiro Harada, Peng-Hui Wang, Ferng-Chun Ke, Wen-Ling Lee and Jiuan-Jiuan Hwang
different promoters confers tissue-specific transcriptional regulation of the Cyp19a1 gene ( Bulun et al . 2003 ). In granulosa cells, this is driven by the ovary-specific PII-promoter that binds cAMP-response element-binding protein (CREB) and nuclear