Autoregulation enables different pathways to control CCAAT/enhancer binding protein β (C/EBPβ) transcription

doi:10.1006/jmbi.2001.4708

Journal of Molecular Biology

Volume 309, Issue 4, 15 June 2001, Pages 855-868

https://doi.org/10.1006/jmbi.2001.4708 Get rights and content

Abstract

CCAAT/enhancer binding protein β (C/EBPβ) also named liver-enriched transcriptional activating protein (LAP) is a member of the C/EBP family of transcription factors and is involved in hepatocyte-specific gene expression and in the process of tissue differentiation. The activity of LAP/C/EBPβ can be regulated at the transcriptional and posttranslational level or by protein-protein interaction with other transcription factors. In this study we show that LAP/C/EBPβ can stimulate its own transcription. Deletion analysis of the rat LAP/C/EBPβ promoter in luciferase reporter gene experiments demonstrated that the region located between nucleotide −121 to −71, comprising two recently characterized cAMP responsive element (CRE)-like elements, is important for autoregulation. Gel shift experiments using oligonucleotides with overlapping point mutations identified the sequence GCAATGA (β-site) adjacent to and partially overlapping the first CRE-like site as core motif for LAP/C/EBPβ binding. Analysis of a mutated β-site in reporter gene experiments showed the functional relevance of this site for autoregulation. The composite C/EBPβ-CRE-element in the promoter enables synergistic activation of transcription by LAP/C/EBPβ and the proteinkinase A (PKA)/cAMP responsive element binding protein (CREB) pathway in a cell-type specific manner. In hepatoma cells nuclear factor kappa B (NF-κB) increased autoregulation and therefore could mediate enhanced activation during inflammatory responses. In summary, our results demonstrated that the assembly of the three binding sites in the promoter and thus the interaction between LAP/C/EBPβ and members of the CREB or NF-κB family allows the control of LAP/C/EBPβ gene transcription as a response to different stimuli in a tissue specific manner.

Introduction

Liver-enriched transcriptional activating protein/CCAAT/enhancer-binding protein β (LAP/C/EBPβ) is a member of the C/EBP family of transcription factors and is an important regulator of hepatocyte-specific gene expression. Specific functions have been described for LAP/C/EBPβ in the regulation of the acute phase response and during liver regeneration. Additionally LAP/C/EBPβ is involved in the process of adipocyte differentiation and metabolism as well as during lymphocyte differentiation.1 The role of LAP/C/EBPβ in the function of other organs is only beginning to become obvious. A normal ovarian physiology is dependent upon LAP/C/EBPβ2 and there is evidence that it may contribute to neuronal differentiation.3

The activity of LAP/C/EBPβ can be regulated at different levels. Specific phosphorylation sites in the LAP/C/EBPβ protein have been shown functionally relevant and demonstrated that posttranslational modifications control the activity of LAP/C/EBPβ.4, 5, 6, 7, 8 Another possibility how LAP/C/EBPβ can be controlled is the interaction with other transcription factors. Protein-protein interaction between LAP/C/EBPβ and glucocorticoid receptors,9 nuclear factor-kappa B (NF-κB),10 the cAMP responsive element-binding protein (CREB)/activating transcriptional factor (ATF) family11 and other C/EBP family members12 have been reported. The resulting heterodimeric complexes have been demonstrated to support, to some extent, altered DNA binding selectivity, and thus different target genes can be modulated. LAP/C/EBPβ can be also controlled on the transcriptional level. This has been demonstrated during the acute-phase response, during liver regeneration and in the course of differentiation processes, when enhanced LAP/C/EBPβ mRNA expression was evident.13 Recently, we identified two CRE-like binding sites in the rat LAP/C/EBPβ promoter which mediate the cAMP response of the gene via the protein kinase A (PKA) pathway in hepatoma and in neuronal cells.14 Additionally the IL-6-dependent increase in LAP/C/EBPβ gene transcription is also mediated by the cAMP responsive element (CRE)-like binding sites through tethering Stat3 to the IL-6-induced CRE-binding protein p68 (M.N. et al., unpublished results).

Autoregulation seems to be a common mechanism for the transcriptional control of all C/EBP family members, whereas the sites involved differ among species. Here, we were interested in analyzing possible autoregulatory mechanisms for the rat LAP/C/EBPβ gene. We define a new binding site in the LAP/C/EBPβ promoter which confers autoregulation. Together with the CRE-like sites targeted by the PKA/CREB pathway, these regions can synergistically activate LAP/C/EBPβ transcription in a cell-type specific manner. Additionally we show that NF-κB increases LAP/C/EBPβ autoregulation in hepatoma cells and, therefore, could mediate its enhanced activity during the inflammatory response. Thus our results indicate that the region between −121 to −71 in the LAP/C/EBPβ promoter can modulate gene transcription dependent on different physiological stimuli.

Section snippets

LAP/C/EBPβ stimulates its own transcription through a region located between −121 and −71 in the promoter region

For several transcriptions factors there is evidence that through transcriptional autoregulatory mechanisms they are able to control their own expression level. Therefore, we were interested to investigate if LAP/C/EBPβ can stimulate its own promoter. The whole 1.4 kb 5′ region of the LAP/C/EBPβ promoter linked to a luciferase reporter gene (LAPPRO 1) was used for this analysis. Cotransfection experiments with a LAP/C/EBPβ expression vector stimulated reporter gene activity (Figure 1(a) and (b))

Discussion

For several transcription factors it has been demonstrated that after an increase in transcription, autoregulatory mechanisms may maintain higher gene transcription by binding of the factor to its own promoter.23 Autoregulation was reported as a main mechanism for C/EBPα activation during adipocyte differentiation24, 25 and to maintain higher C/EBPδ expression levels after a Stat3-dependent increase in gene transcription during the acute phase response.26 Different autoregulatory mechanisms

LAP/C/EBPβ promoter constructs

The LAPPRO 1, 5, 8 and 9 constructs corresponding to increasing deletions in the 5′ flanking region of the LAP/C/EBPβ open reading frame linked to a luciferase reporter gene were as described.14 The LAPPRO 8 MUTCRE1 construct carries mutations in the first CRE-like site (−109 to −107=ACG→GTT) and the LAPPRO 8 MUTCRE2 construct carries mutations in the second CRE-like site (−65 to −61=TGACG→GATCC) as described elsewhere.14 The mutations −113 to −111 (AAT→GGA) and −65 to −61 (TGACG→GATCC) were

Acknowledgements

This work was supported by a grant of the Deutsche Forschungsgemeinschaft DFG Tr 285 4-3.

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CCAAT/enhancer binding protein (C/EBP) β and C/EBPδ are transcription factors of the basic-leucine zipper class which share phylogenetic, structural and functional features. In this review we first describe in depth their basic molecular biology which includes fascinating aspects such as the regulated use of alternative initiation codons in the C/EBPβ mRNA. The physical interactions with multiple transcription factors which greatly opens the number of potentially regulated genes or the presence of at least five different types of post-translational modifications are also remarkable molecular mechanisms that modulate C/EBPβ and C/EBPδ function. In the second part, we review the present knowledge on the localization, expression changes and physiological roles of C/EBPβ and C/EBPδ in neurons, astrocytes and microglia. We conclude that C/EBPβ and C/EBPδ share two unique features related to their role in the CNS: whereas in neurons they participate in memory formation and synaptic plasticity, in glial cells they regulate the pro-inflammatory program. Because of their role in neuroinflammation, C/EBPβ and C/EBPδ in microglia are potential targets for treatment of neurodegenerative disorders. Any strategy to reduce C/EBPβ and C/EBPδ activity in neuroinflammation needs to take into account its potential side-effects in neurons. Therefore, cell-specific treatments will be required for the successful application of this strategy.
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While ROS-dependent C/EBPβ up-regulation, which was only evident in TAK1/p38 MAPK signaling-incompetent VSMCs, primarily resulted from ROS-dependent NRF2 up-regulation, how ROS-independent C/EBPβ up-regulation was affected by IKKβ signaling remains obscure. Though IKKβ may promote C/EBPβ autoregulation through activating NF-κB [21], SN50, a cell-permeable peptide that inhibits NF-κB nuclear translocation, did not significantly inhibit expression of C/EBPβ and SDF-1 (data not shown). As cAMP-dependent protein kinase signaling, which also affects C/EBPβ expression [22], can also be regulated by IKKβ in an NF-κB-independent manner [23], whether similar NF-κB-independent effects of IKKβ contribute to IL-1α-induced expression of C/EBPβ and SDF-1 in VSMCs deserves further investigation.
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¹: Edited by M. Yaniv

²: Present address: M. Niehof, Fraunhofer-Institut, D-30625 Hannover, Germany.

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Journal of Molecular Biology

Regular articleAutoregulation enables different pathways to control CCAAT/enhancer binding protein β (C/EBPβ) transcription1

Abstract

Introduction

Section snippets

LAP/C/EBPβ stimulates its own transcription through a region located between −121 and −71 in the promoter region

Discussion

LAP/C/EBPβ promoter constructs

Acknowledgements

J. Biol. Chem.

Mol. Cell

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

J. Biol. Chem.

Int. J. Biochem. Cell Biol.

Trends Genet.

Biochem. Biophys. Res. Commun.

Biochim. Biophys. Acta

J. Biol. Chem.

Advan. Immunol.

FEBS Letters

J. Biol. Chem.

J. Biol. Chem.

An essential role for C/EBPβ in female reproduction

Genes Dev.

CCCAAT/enhancer binding protein beta is a neuronal transcriptional regulator activated by nerve growth factor receptor signaling

J. Neurochem.

cAMP stimulates the C/EBP-related transcription factor rNFIL-6 to translocate to the nucleus and induce c-fos transcription

Genes Dev.

Phosphorylation at threonine 235 by ras-dependent mitogen activated protein cascade is essential for transcription factor NF-IL6

Proc. Natl Acad. Sci. USA

Transactivation by LAP/NF-IL6 is enhanced by phosphorylation of its activation domain

Nature

Calcium-regulated phosphorylation within the leucine zipper of C/EBPβ

Science

A nuclear factor for interleukin-6 expression (NF-IL6) and the glucocorticoid receptor synergistically activate transcription of the rat alpha 1-acid glycoprotein gene via direct protein-protein interaction

Mol. Cell Biol.

The p50 subunit of NF-kappa B associates with the NF-IL6 transcription factor

Proc. Natl Acad. Sci. USA

C/ATF, a member of the activating transcription factor family of DNA-binding proteins, dimerizes with CART/enhancer-binding proteins and directs their binding to cAMP response elements

Proc. Natl Acad. Sci. USA

Regular article
Autoregulation enables different pathways to control CCAAT/enhancer binding protein β (C/EBPβ) transcription¹