AIM: To obtain human esophageal cancer cell EC9706 stably expressed epithelial membrane protein-1 (EMP-1) with integrated eukaryotic plasmid harboring the open reading frame (ORF) of human EMP-1, and then to study the mechanism by which EMP-1 exerts its diverse cellular action on cell proliferation and altered gene profile by exploring the effect of EMP-1.

METHODS: The authors first constructed pcDNA3.1/myc-his expression vector harboring the ORF of EMP-1 and then transfected it into human esophageal carcinoma cell line EC9706. The positive clones were analyzed by Western blot and RT-PCR. Moreover, the cell growth curve was observed and the cell cycle was checked by FACS technique. Using cDNA microarray technology, the authors compared the gene expression pattern in positive clones with control. To confirm the gene expression profile, semi-quantitative RT-PCR was carried out for 4 of the randomly picked differentially expressed genes. For those differentially expressed genes, classification was performed according to their function and cellular component.

RESULTS: Human EMP-1 gene can be stably expressed in EC9706 cell line transfected with human EMP-1. The authors found the cell growth decreased, among which S phase was arrested and G1 phase was prolonged in the transfected positive clones. By cDNA microarray analysis, 35 genes showed an over 2.0 fold change in expression level after transfection, with 28 genes being consistently up-regulated and 7 genes being down-regulated. Among the classified genes, almost half of the induced genes (13 out of 28 genes) were related to cell signaling, cell communication and particularly to adhesion.

CONCLUSION: Overexpression of human EMP-1 gene can inhibit the proliferation of EC9706 cell with S phase arrested and G1 phase prolonged. The cDNA microarray analysis suggested that EMP-1 may be one of regulators involved in cell signaling, cell communication and adhesion regulators.

EMP-1 is a member of the PMP22 family with the similarity in structure. Since EMP-1 was first found by Taylor, it has been isolated independently from human, mouse and rabbit and received many different designations, such as TMP (tumor membrane Protein), PAP (Progression Associated Protein), CL-20 and B4B[1]. All tissues expressing EMP-1 mRNA contain 2.76-kb EMP-1 transcripts. In some regions of the gastrointestinal tract, including the fundus, ileum, cecum, and colon, however, additional transcripts of approximately 1.7 kb hybridize with the EMP-1 cDNA[2] .The 2.76-kb EMP-1 cDNA contains five exons about 0.2kb, 0.12kb, 0.1kb, 0.14kb, and 2.2 kb and four introns about 15kb, 1.9kb, 0.1kb, and 0.7 kb in length respectively. EMP-1 has been mapped to chromosome 12p12 by fluorescence in situ hybridization[3]. EMP-1 is encoded by a single-copy gene with the positions of introns exactly conserved between EMP-1 and PMP22, corroborating the hypothesis that EMP-1 belongs to the PMP22 family[4]. EMP-1 transcript is expressed at high levels in heart, placenta, lung, skeletal muscle, kidney, spleen, colon prostate, ovary, testicle, small intestine and thymus in human[5].

EMP-1 was selected from a series of differential expressed genes obtained from cDNA microarray analysis of expression profiles of esophageal cancer in our previous work. EMP-1 expression was 6 fold down-regulated in esophageal cancer lower than in normal tissue. EMP-1 is highly up-regulated during squamous cell differentiation and in certain tumors, and a role in tumorigenesis has been proposed[6]. Moreover, The overexpression of PMP22 leads to an apoptotic-like phenotype in NIH3T3 growing cells[7] and delays serum-forksolin-stimulated entry of resting Schwann cells from G1 into the S+G2/M phase in Schwann cell[8]. Transient expression of EMP-1 specifically inhibited cellular proliferation by more than 50%[9]. Preliminary data suggested that EMP-1 was involved in growth control in esophageal cancer cell line EC9706. However, whether there is a similar effect of EMP-1 expression on the cell cycle of epithelial cells remains to be determined and little is known about the function of EMP-1 in growth control in esophageal cancer cell line EC9706.

To elucidate the effect of EMP-1 on EC9706 cell, the open reading frame (ORF) of human EMP-1 was cloned into pcDNA3.1/myc-his, a eukaryotic expression vector. EC9706 was transfected with the integrated plasmid containing EMP-1 to enforce expression of the exogenous EMP-1. Western blotting and RT-PCR were used to analyze positive clones. The cell growth curve was observed and the cell cycle was checked by FACS method. However, the mechanism by which EMP-1 may exert its activity remains unclear. Because the differentiation of mammalian cells is associated with changes in gene expression that is primarily controlled at the level of transcription, we tested the expression alteration with cDNA microarray technology to address the question of which genes are influenced by EMP-1 gene overexpression.

In our previous work on the expression profile of esophageal cancer, we found that the expression of EMP-1 was 6 fold down-regulated in esophageal cancer than that in its adjacent normal mucosa. This study was designed to determine the diverse cellular action of EMP-1 by enforcing its expression in EC9706. Human EMP-1 gene can be stably expressed by EC9706 cell line transfacted with human EMP-1. As a result, the cell proliferation was inhibited, S phase was arrested and G1 phase was prolonged after EMP-1 overexpresssion in EC9706 cells, which is similar to the action of PMP22 in Schwann cells[8]. We then analyzed EMP-1 induced gene expression using Atlas^TM human cancer cDNA expression microarray composed of 588 genes, 35 genes showed an over 2.0- fold change in expression level, in which 28 genes were consistently up-regulated and 7 genes were down-regulated. Among the classified genes, almost half of the induced genes (13 out of 28 genes) were related to cell signaling, communication and adhesion, which indicated that EMP-1 may be one of the cell signaling, communication and adhesion regulators (Table 2).

cDNA microarray analysis offers the opportunity to monitor changes in gene expression across the entire set of expressed genes in cells. These 588 known genes are classified on the basis of the biological function of the encoded protein, using a modified version of a previously established classification scheme[11,12]. The classification scheme was composed of six major functional categories and several minor functional categories within the major categories. As shown in Table 2, 34 genes were classified as known function genes, and one gene was categorized as unclassified.

Interestingly, the largest categories (13 out of 28 genes) of EMP-1-induced genes are those involved in cell signaling (Table 1), cell adhesion and cell-cell communication, which included integrin beta 7 (ITGB7), integrin beta 8 (ITGB8) and cadherin 5 (CDH5). These results indicated that EMP-1 might be related with cell adhesion and cell-cell communication.

Cadherin-5 and the alpha E beta 7 integrin mediated heterotypic adhesive interactions between epithelial cells and intraepithelial lymphocytes in vitro[13]. Integrin beta 7 (ITGB7) and integrin beta 8 (ITGB8) are heterodimeric (alpha/beta) transmembrane receptors for extracellular matrix (ECM) ligands. The beta subunit of integrins are considered important for regulation of stimulated cell adhesion and adhesion-dependent signal transduction. Through interactions with molecular partners at cell junctions, they provide a connection between the ECM and the cytoskeleton and regulate many aspects of cell behaviors. Integrins play an important role in lymphocyte adhesion to cellular and extracellular components of their microenvironment[14]. Cadherin 5 was also induced by EMP-1 in our microarray analysis. It is generally accepted that cadherins are a group of cell adhesion molecules located at intercellular junctions, and they play an important role in embryogenesis and morphogenesis in animals and humans due to their adhesive and cell-signaling functions. Disturbances of the expression or function of cadherins and their associated proteins are crucial for the initiation and development of many pathological states. Cadherin 5 is an epithelium-specific cadherin that is required for the development and maintenance of the normal function of all epithelial cells in tissues. The loss or down-regulation of cadherin 5 is a key event in the process of tumour invasion and metastasis[15] Cadherin 5 has the major role in intercellular adhesion in esophageal mucosa[16]. Appropriate cadherin expression reflects the differentiation of squamous cell carcinoma[17]. These results support the putative function of EMP-1, which was a potential maker of differentiation[18] and was related to cell proliferation, differentiation, regulation and cell death[3].

In addition, the second largest categories of EMP-1-induced genes were those involved in cell division, Such as cyclin-dependent kinase 4 inhibitor 2D, DNA damage-inducible transcript 3, and retinoic acid receptor beta. The up-regulated cyclin-dependent kinase 4 inhibitor 2D and the down-regulated genes in this categories including cell division cycle 25 homolog A[19] and cyclin D2 (CCND2) and cyclin D3 (CCND3)[20] affect the cell cycle collaboratively. These support our finding that the inhibition of cell proliferation after overexpression of EMP-1 associates with the induction of S phase arrested and the prolonged G1 phase with flow cytometric assay.

Retinoic acid receptors beta (RAR-β) is another up-regulated gene induced by EMP-1. It is well established that retinoids can modulate epithelial cell growth, differentiation, and apoptosis in vitro and in vivo by binding to specific nuclear retinoid receptors, which include RAR-β[21]. Retinoids can prevent abnormal squamous cell differentiation in nonkeratinizing tissues physiologically. Retinoids can also reverse squamous cell metaplasia, which develops during vitamin A deficiency[22]. The expression of RAR-beta varied along with the differentiation level of esophageal cancer. RAR-beta mRNA was expressed in 62.7%(42/67), 55.1%(43/78) and 29.2%(7/24) of well, moderated and poorly differentiated SSCs, respectively[23]. Retinoic acid, which inhibits squamous cell differentiation with RAR-beta involved in, represses EMP-1 expression in normal human bronchial epithelial cells[4]. EMP-1 is one of down-regulation of a cluster of squamous cell differentiation marker genes[18] and RAR-β may be involved in this process.

Some genes associated with the intracellular signaling pathway, such as dishevelled homolog 1-like protein and ras associated with diabetes protein 1, were induced by EMP-1 overexpression (Table 1).

A study addressing the relationship between EMP-1 and these cell adhesion and cell-cell communication proteins has not, to our knowledge, previously been reported. The presumed protein structure of EMP-1 also supports its function in cell signaling, cell adhesion and communication. The predicted EMP-1 polypeptide of 157 amino acid residues has a calculated molecular weight of approximately 18 kDa, and bioinformatics analysis reveals that amino acid residues 1-28, 64-89, 95-117, and 134-157 represent four hydrophobic, potentially membrane-spanning domains. Computer-predicted structural domains of EMP-1 are partially mirrored by the exon/intron structure of EMP-1. Most interestingly, exon 4, which covers the potential second transmembrane domain, a small intracellular loop, and half of the third transmembrane domain, encodes the most highly conserved regions between the EMP-1 and PMP22 proteins indicating some shared functional significance for this module in the PMP22 family. Because of the small size of the intracellular loops, it appears unlikely that they are involved in specific interaction with intracellular proteins and therefore in intracellular signaling. In contrast, the extracellular loops would be able to interact with other molecules and could have functional significance. Furthermore, the first hydrophilic region of PMP22/EMPs, between the first and second hydrophobic domains, forms an extracellular loop that contains one or more consensus sequences that can be N-linked glycosylated in vitro, a structure which has been implicated in cell-cell recognition and adhesion processes[4,24].

In conclusion, the study of the gene expression changes sustains cellular finding that the inhibition of cell proliferation after enforcing expression of EMP-1 associates with the induction of S phase shorten and the prolonged G1 phase. The expression profile and localization of EMP-1 suggests a role in cell signaling, adhesion and communication. This is the first demonstration of global gene expression analysis of esophageal cancer cell line EC9706 transfectant with EMP-1, and these results provides a new insight in the study of the relationship between EMP-1 and esophageal cancer.