Elsevier

Journal of Hepatology

Volume 44, Issue 4, April 2006, Pages 758-767
Journal of Hepatology

Eve-3: A liver enriched suppressor of Ras/MAPK signaling

https://doi.org/10.1016/j.jhep.2005.10.031Get rights and content

Background/Aims

The developed liver is able to tightly control cellular proliferation, rapidly switching from quiescence to growth in response to specific stimuli. This suggests that growth inhibitors may be involved in the control of liver growth. We analyzed the role of the Spred-family of growth inhibitors in the liver.

Methods

We screened human EST databases for Spred-related sequences. Clones were isolated, sequenced, epitope-tagged and expressed. Subcellular localization of clones were determined and their effects on cellular signaling pathways analysed using specific antibodies. Cell cycle progression assays and protein interaction studies were initiated. Organ distribution of transcripts and their expression throughout liver development and in primary hepatocytes were recorded.

Results

We have identified a new, liver-restricted protein, Eve-3, containing a single Ena Vasp homology (EVH1) domain that can potently block activation of the Ras/MAPK pathway. Eve-3 is specific in inhibiting the Ras/MAPK pathway. Eve-3 can block serum-mediated cell cycle progression and its expression is highly regulated during liver development.

Conclusions

The liver is the only organ that can regulate its growth and mass. Eve-3 may act as an inhibitor of proliferation pathways in the mature liver and be involved in modulating the unique regenerative capacity of this organ.

Introduction

The process of normal development depends upon a coordinated and tightly regulated response to growth factors that stimulate cellular proliferation and differentiation. The intracellular Ras/MAPK cascade amplifies the input growth factor signal, and activation of this pathway is essential for cell cycle progression and cell growth. Recently two new classes of negative regulatory proteins, Sprouty [1], [2] and Spred [3], which can antagonize mitogenic signaling by growth factor receptors have been characterized. Spred and Sprouty proteins share a common C-terminal region, the Sprouty domain, that appears to target the proteins to the cell membrane and is necessary for inhibitory function [3], [4]

The mature hepatocyte is a highly differentiated cell that performs a plethora of metabolic functions and is noted for its quiescence or minimal replicative activity [5], [6]. Strikingly, however, in response to trauma or toxic insult, differentiated hepatocytes can be mobilised to reenter the cell cycle and undergo cell division. After 70% partial hepatectomy, in which the large and the median lobes of the rodent liver are removed, more than 95% of mature hepatocytes exit the quiescent G0 phase and proceed through the G1-S transition point of the cell cycle [6]. The proliferative events following partial hepatectomy in the adult, are mirrored in reverse during liver development, with embryonic and early postnatal hepatocytes having a high proliferative index, until the fourth postnatal week when there is a sharp drop in hepatocyte proliferation [7].

The major intracellular signal transduction pathway controlling cell growth is the Ras/mitogen activated protein (MAP) kinase cascade [8]. Ras, activated by growth factor receptors, activates the Raf protein kinase. Raf in turn phosphorylates and activates MEK, which in turn phosphorylates and activates MAP kinases including ERK1 and ERK2. Overactivation of the Ras/Raf/MAPK pathway has also been implicated in the development and progression of human carcinoma, including hepatocellular carcinoma. Based on sequence homology analysis with Spred proteins we have identified a new protein, Eve-3, containing a single Ena Vasp homology (EVH1) domain that can potently block activation of the Ras/MAPK pathway. Eve-3 expression is restricted to the liver and is tightly regulated during development suggesting that Eve-3 may play a role in maintaining liver quiescence.

Section snippets

Cloning of Eve-3/Spred-3 and cell transfection

Eve-3 and Spred-3 expression vectors were constructed from two EST clones, GenBank accession numbers: BF983829 and BF409205 via PCR (Platinum Pfx polymerase: Invitrogen) according to the manufacturer's conditions. Non-overlapping regions were filled in by PCR using sequence information from ESTs and the genomic eve-3 gene obtained from the public and Celera genomic databases. Constructs were fully sequenced prior to use. hEve-3 and hSpred-3 were N-terminal Flag- and HA-tagged and cloned into

Isolation of a new, liver-restricted Spred protein family member

We screened the human EST database for cDNAs homologous to the Spred proteins and found one initial EST (GenBank no.: BF983829) with similarity to Spred proteins. Through genomic analysis we determined that the cDNA encoded by the EST mapped to the Spred-3 locus. Surprisingly, this gene encodes two distinct proteins, the first containing only an N-terminal Ena Vasp Homology 1 (EVH1) domain and a unique C-terminal segment. We termed this protein Eve-3 (EVH1 enhanced). A second, larger protein,

Discussion

Eve-3 is a distinct protein encoded by the Spred-3 gene and expressed in a liver restricted pattern. Eve-3 comprises an N-terminal EVH1 domain and a unique C-terminal region encoded by exon 5, which possesses an in frame stop codon. Sprouty domain deletion mutants of mouse Spred-1 and Spred-2, similar in domain structure to Eve-3, were able to augment cell differentiation and growth factor induced activation of ERK2 [3] suggesting that these constructs have dominant negative capabilities. In

Acknowledgements

This work was supported by Victor Hurley Medical Research Fund, RMH, the Royal Australasian College of Surgeons and the Melville Hughes Scholarship, NHMRC, Australia, the RMH Neuroscience Foundation, Australia, the Roche Foundation, Switzerland and a donation from James and Linda Wang. We thank Dr Marie Bogoyevitch (University of W.A. Australia) for the p38/JNK constructs and Prof. Rony Seger (Weizmann Institute, Israel) for the GFP-ERK2 construct.

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