Cloning And Functional Characterization Of A Novel Mouse Molecule MPEBP4

The phosphatidylethanolamine-binding protein (PEBP) family consists of a number of 21-23-kDa basic proteins, first identified in bovine brain, with preferential in vitro affinity for phosphatidylethanolamine (PE), a component of the cell membrane. This family is an evolutionarily conserved group found in species of flowering plants (Antirrhinum), parasites (Plasmodium falciparium), nematodes (Toxocara canis), insects (Drosophila melanogaster), and mammals including cattle, monkeys, and humans. A number of functions have been suggested for the mammalian PEBP proteins, including lipid binding and inhibition of serine proteases. These proteins can also act as precursors for a bioactive peptide HCNP (hippocampal cholinergic neurostimulating peptide), which is important in hippocampus development. Human PEBP1 (hPEBPl, also called Raf kinase inhibitory protein of RKIP) acts as a suppressor of Raf-1 kinase activity and mitogen-activated protein kinase (MAPK) signaling in fibrobblasts via its ability to sequester and inactivate Raf-1 and MEK1. Both Raf-1 and MEK bind to the highly conserved PE-binding domain of hPEBP4;so, hPEBP4 induces dissociation of Raf-1 MEK complexes and behaves as a competitive inhibitor of MEK phosphorylation. Some studies also show that RKIP might act as a suppressor of prostate cancer metastasis.hPEBP4, a novel member of phosphatidylethanolamine-binding protein (PEBP) family, was isolated from the human BMSC cDNA library by large-scale random sequencing in our lab. We demonstrate that hPEBP4 is highly expressed in human breast cancer and acts as anti-apoptotic molecule. Once hPEBP4 was silenced in breast cancer cells, the breast cancer cells became more sensitive to TNFalpha-induced cellular apoptosis, indicating the hPEBP4 may be a candadite target gene for breast cancer therapeutics. On the basis of previous studies abouthPEBP4, we want to identify the mouse homolog of hPEBP4 and characterize its functions. Here we report the cloning and functional characterization of mPEBP4.Part I. Cloning, sequence analysis, expression pattern and cytolocalization of mPEBP4Through homology analysis in the PEBP family and bioinformatics search, we found an unknown protein from Mus musculus (GenBank Accession Number AK006964), which contains a PE-binding domain, in this case between amino acids 106-213. According to the report by O'Bryan and colleagues, previously identified PEBPs can be grouped into three subfamilies;the majority of differences between these subfamilies are in the N-terminal residues (those preceding the PE-binding domain), especially the first 10 amino acids. Members of the three subfamilies share similar overall features, including length (each being about 190 residues) and amino acid composition in functional domains. In contrast, hPEBP4 and the unnamed mouse protein BAB24810 differ in these aspects. They are both more than 220 residues in length, and contain at least 2 insertions and 1 deletion in their protein sequence, when compared to proteins belonging to the three existing subfamilies. So, we named BAB24810 as mPEBP4. The residues, which were previously demonstrated to be conserved among all members of the family (Proll9x Aspl20> Prol22> Hisl34 and Argl719), were also found in mPEBP4. These residues are thought to be involved in determining the local structure of a biologically important ligand-binding site in PEBPs. The two main regions of high sequence conservation occurred between residues 113-139 and residues 163-175 (based on mPEBP4 numbering). Residues 118-120 with 113-123 and 122 forming an Asp-Pro-Asp-x-Pro motif that is universally conserved in all family members. The 163-175 conserved region formed part of the second side of the binding pocket. These structural features suggested that mPEBP4 might possess PE binding properties similar to those seen in other PEBPs.To our interest, RT-PCR analysis revealed that mPEBP4 mRNA was specifically expressed in eyes tissue. We did not detect its expression in other tissues such as bonemarrow, uterus, prostate, smooth muscle, stomach, liver, brain, hippocampus (so, we cloned the full-length cDNA of mPEBP4 from the mice eyes tissue). We also examined the expression pattern of hPEBP4 mRNA in a variety of tumor cells and freshly isolated cells including mouse dendritic cells, L929 (fibroblast), B16F10 (melanoma), 3LL (lung carcinoma), 4T1 (breast carcinoma), CT26 (colon carcinoma), A20 (B cell lymphoma), and found that mPEBP4 was only expressed weakly in NIH3T3 cells. Following Epirubicin stimulation, mPEBP4 expression increased in the 4T1 cells, which do not normally express mPEBP4, reached a maximum at 24 hr, and then decreasing. Furthermore, the treatment of TNFa or TRAIL could not induce mPEBP4 expression in breast carcinoma 4T1 cells, which different to the inducible expression pattern of hPEPB4 in human breast cancer cells upon TNFa or TRAIL treatment.The full ORF of mPEBP4 was cloned from mouse eyes cDNA which encoded a 242-amino acid protein. In order to investigate the cytolocalization of mPEBP4, we constructed the expression vector of full-length mPEBP4 fused to GFP protein in C-terminal fusion (mPEBP4-GFP) and transiently transfected it into the HEK293 cells. Fluorescence confocal analysis showed that mPEBP4-GFP was partly co-localized with endoplasmic reticulum and Golgi's apparatus.Part II. Functional characterization of mPEBP4Metastasis is the main cause of death for most cancer patients. The metastatic process of cancer cell involves a complex cascade of events. In brief, a metastatic cancer cell must escape from the primary tumor, enter the circulation, arrest in the microcirculation, invade a different tissue compartment, and then grow at that secondary site. It has been reported that RKIP, a member of the PEBP family, acts as a suppressor of prostate cancer metastasis. Therefore, we investigated whether mPEBP4 can affect the migration and invasion of mouse breast cancer cells 4T1. Using scratch method and Transwell assay, we found that mPEBP4 overexpression could promote the migration and invasion of 4T1 cells. It's well known that MAPKand PI3K/Akt pathways are important signaling pathways which account for the metabolism, apoptosis and proliferation of cells, and some studies show that they are required for cell migration. So, we then examined whether these signal pathways are involved in the promoting effect of mPEBP4 on the migration and invasion of 4T1 cells. We found overexpression of mPEBP4 inhibited serum-induced ERKl/2 and JNK activation but had no effect on p38 and Akt activation. Simultaneously, we examined the expression of some molecules associated with cellular migration, such as COX-2, MMP-3/9, TGF- 3 , CCR7 and CXCR4. Our findings showed that only COX-2 mRNA expression was up-regulated in mPEBP4-B-overexpressing 4T1 cells. These results suggested that mPEBP4 might promote the migration and invasion of mouse breast cancer cells 4T1 by inhibiting the activation of ERKl/2 and JNK and up-regulating the expression of COX-2.Since our previous study showed that hPEBP4 could inhibit TNFa and TRAIL-induced apoptosis, we also examined if mPEBP4 plays a role in apoptosis. Since Epirubicin plays an important role in treating breast carcinoma as one kind of apoptosis-inducing agent, we used it to induce apoptosis of mouse breast cacinoma cells 4T1. Our findings showed that mPEBP4 could inhibit Epirubicin-induced apoptosis. MAPK pathway is an improtant signaling pathway which account for the apoptosis resistance, so we then examined the effect of mPEBP4 on this pathway. When compared with mock transfectants or wildtype, mPEBP4-B transfectants showed more ERKl/2 and JNK activation upon Epirubicin stimulation. But the exact mechanisms by which mPEBP4 acts as anti-apoptotic molecule remain unkown, and further investigation should be carried out.In conclusion, we have cloned and functional characterized mPEBP4, a novel member of the PEBP family, which showed similar structural features to hPEBP4. We found that mPEBP4 could promote the migration and invasion of breast cancer cells 4T1 via inhibiting ERKl/2 and JNK activation and up-regulating the expression of COX-2. mPEBP4 also plays a negative role in Epirubicin-induced apoptosis probably by promoting activation of ERKl/2 and JNK. Considering mPEBP4 not onlypromotes survival of breast cancer cell, but also promotes its migration and invasion, mPEBP4 may be a new molecule needed to be further investigated.