Elsevier

Journal of Hepatology

Volume 54, Issue 6, June 2011, Pages 1185-1194
Journal of Hepatology

Research Article
Characterization of the transcriptional signature of C/EBPbeta isoforms (LAP/LIP) in Hep3B cells: Implication of LIP in pro-survival functions

This paper is dedicated to the memory of Doctor Jean-Philippe Salier who died while the work was in progress.
https://doi.org/10.1016/j.jhep.2010.09.021Get rights and content

Background & Aims

C/EBPbeta is an important mediator of several cellular processes, such as differentiation, proliferation, and survival of hepatic cells. However, a complete catalog of the targets of C/EBPbeta or the mechanism by which this transcription factor regulates certain liver-dependent pathways has not been clearly determined. Two major natural isoforms of this transcription factor exist: the liver-enriched activating protein (LAP) and the liver-enriched inhibitory protein (LIP), a functional LAP antagonist. In this study, we used the opposing transcriptional effects driven by LAP and LIP to determine the genuine C/EBPbeta molecular signature in the Hep3B human hepatoma cell line. We subsequently investigated the role of each of the LAP and LIP isoforms in drug-induced Hep3B cell death.

Methods

We engineered Hep3B cells with regulated LAP or LIP expression using the Tet-off expression system. The genes that showed inverse regulation by LAP and LIP were identified by cDNA array analysis. The cohort of direct-C/EBPbeta-targets was distinguished from indirect-targets by ChIP-on-chip analysis.

Results

We characterized 676 genes by this approach. Among these genes, 39 are novel direct targets of C/EBPbeta. Eleven of these new direct targets are involved in cell survival, suggesting critical roles for LAP/LIP isoforms in this cellular process. Therefore, we examined the effects of LAP and LIP over-expression on cell survival. We show that LIP promotes survival in staurosporine- or taxol-induced Hep3B cell death.

Conclusions

Our study provides new molecular and cellular insights into the role of C/EBPbeta in cells of hepatic origin.

Introduction

CCAAT/enhancer-binding proteins (C/EBPs) are a family of leucine zipper transcription factors involved in the regulation of various functions in multiple tissues [1], [2]. One member of this family, C/EBPbeta, is a liver-enriched transcription factor. It plays critical roles in the inflammatory response, tumorigenesis, differentiation, proliferation, and survival of hepatocytes by regulating the expression of liver-specific genes [3], [4].

A variety of strategies have been used to determine C/EBPbeta targets in hepatic cells such as modulation of C/EBPbeta expression (knockdown or over-expression) or computational prediction of C/EBPbeta binding site locations. However, a full ensemble of C/EBPbeta targets or the mechanisms by which this transcription factor regulates certain liver-dependant pathways has not been clearly determined. The major reason for this is the increasing complexity by which C/EBPbeta affects gene expression. The absence of C/EBPbeta may be compensated for by other C/EBP proteins [5], [6]. Additionally, the transactivating capacity of this transcription factor is regulated by several types of post-translational modifications (phosphorylation, sumoylation, etc.) or by dimerization with other DNA binding proteins [2], [3], [7], [8]. The intronless CEBPB gene is a gene known to generate a single mRNA that is alternatively translated into three isoforms, the 38 and 35 kDa liver-enriched activator proteins (LAP∗ and LAP, respectively), and the 20 kDa liver-enriched inhibitory protein (LIP). LAP and LIP are the most highly expressed isoforms. LAP contains both the transactivation and specific DNA binding domains, while only the latter is conserved in LIP (Supplementary Fig. 1) [9]. Thus, LIP can act as a dominant-negative inhibitor of LAP-mediated gene expression [9]. Indeed, growing evidence has shown that LAP and LIP are involved in the opposing transcriptional regulation of genes coding for proliferation proteins during liver regeneration and for genes coding for acute phase proteins during inflammation [10], [11], [12].

In the present study, we identified new relevant targets of C/EBPbeta using engineered Hep3B cells with Tet-off inducible LAP or LIP proteins. The expression profiling of these cells enabled the identification of genes whose expression was differentially regulated by LAP and LIP (i.e. a genuine C/EBPbeta molecular signature). Among these genes, positive hits that contained an occupied C/EBPbeta binding site in their upstream regions (i.e. putative direct-targets of C/EBPbeta) were further identified by ChIP-on-chip analysis. We characterized 11 new direct C/EBPbeta transcriptional targets coding for proteins involved in the apoptosis processes. We further confirmed the implication of LAP in the down-regulation of hepatoma cell proliferation. Moreover, we evaluated the function of LAP and LIP isoforms in cell death.

The identification of biologically relevant direct and indirect transcriptional-targets of C/EBPbeta has contributed to the increase in knowledge on the diverse functions controlled by this transcription factor in cells of hepatic origin. Moreover, we bring to light new findings on the potential role of LIP in cell death.

Section snippets

Establishment of stable Hep3B hepatoma cell clones expressing each of the C/EBPbeta isoforms (LAP or LIP)

We established stable Hep3B hepatoma cells that produced in a doxycycline-controlled manner the M2-flag flagged LAP (35 kd) or LIP (20 kd) using the Tet-off expression system (Clontech, Ozyme). For each isoform, two clones, referred to as Hep3BLAP-I, Hep3BLAP-II, Hep3BLIP-I, and Hep3BLIP-II, were made (see Supplementary materials). All mRNA levels were determined using Quantitative Reverse Transcription Polymerase Chain Reaction (Q-RT-PCR) analysis, and protein levels were determined by Western

Hep3B human hepatoma cell clones over-expressing either LAP or LIP proteins

To better understand the role of C/EBPbeta in cells of hepatic origin, we intended to characterize the biological targets and cellular functions under the differential control of LAP and LIP.

We engineered hepatoma cell lines over-expressing specifically and in a controlled manner either LAP or LIP using the Tet-off system. Our approach using this ex-vivo model-system is not physiologically relevant. However, it provides a valuable model to better understand the role that these proteins have in

Implication of LAP and LIP in the regulation of liver specific-genes

To dissect the molecular role of C/EBPbeta in hepatic cells, we have identified the genes that are under the differential control of LAP or LIP. Our strategy was to engineer a human hepatoma cell line over-expressing LAP or LIP in a controlled manner. It was crucial to choose the appropriate cell line that would exhibit the largest change in gene expression under control of the C/EBPbeta isoforms. We showed that the Hep3B cell line was very relevant for such studies.

In vivo or in vitro systems

Conclusions

In summary, we have identified a specific gene signature controlled by C/EBPbeta in hepatic cells, i.e. the genes under differential LAP and LIP regulations. We have provided new evidence for implication of LAP and LIP in the regulation of liver-specific genes. In addition, we have provided a functional analysis showing that LAP and LIP may regulate cell proliferation and cell death, respectively. Because the over-expression of exogenous C/EBPbeta may induce non-specific effects, additional in

Conflict of interest

The authors who have taken part in this study declared that they do not have anything to disclose regarding funding or conflict of interest with respect to this manuscript.

Acknowledgments

We thank Dr. Papaconstantinou for plasmids; Dr. Perret for her advice and gift of HuH6 cells; J. Bou for her brilliant technical assistance; Dr. Tron for his advice. This work was supported in part by grants from Association de Recherche sur le Cancer and Ligue contre le Cancer.

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    These authors contributed equally to the work.

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