Journal of Molecular Biology
Regular articleAutoregulation enables different pathways to control CCAAT/enhancer binding protein β (C/EBPβ) transcription1
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element (CRE)-like binding sites through tethering Stat3 to the IL-6-induced CRE-binding protein p68 (M.N. et al., unpublished results).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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2015, Biochemical and Biophysical Research CommunicationsCitation Excerpt :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.