Study Of Neutrophil Gelatinase-associated Lipocalin Receptor In Esophageal Carcinoma Cells

Recent studies suggest that NGAL is a novel iron transporter with functions distinct from that of transferrin by binding a specific cellular receptor and mediate a new iron delivery pathway. Our previous work have identified that NGAL was overexpressed during the progression from normal epithelium to invasive ESCC. Further research demonstrated that expression of NGAL was closely associated with tumor invasion and differentiation degree of ESCC. However, a clear explanation for the overexpression of NGAL has been lacking. In the present study, We found that NGAL mediated iron delivery process was present in the esophageal inflammatory epithelium and carcinoma tissues and the expression of NGAL in the esophageal carcinoma cells could be induced following stimulation by inflammatory factors. According to the study, we suppose that the delivery of vast irons to cells, which was mediated by NGAL protein, would add to the growth potential of the tumor combined with a grave free radical reaction and gene mutation thus promoting malignancy in esophageal carcinoma. In the current item, we analyzed the expression of NGAL receptor in esophageal carcinoma cells and identified a novel NgalR spliced variant designated as NgalR-3. When expressed in a heterologous system, the protein produced from this novel spliced variant exhibits the biochemical characteristics of interaction and co-localization with NGAL protein in vivo. This new finding suggests that NgalR-3 may act as a potential NGAL receptor and play a role in NGAL mediated iron transporting process in the esophageal carcinoma.Methods:1. NgalR isoforms mRNA level were detected in several esophageal carcinoma cells, esophageal carcinoma and adjacent normal mucosa tissues.2. Human NgalRs variant cDNA was cloned from esophageal carcinoma EC18 cells by RT-PCR. The amplified PCR fragment was verified by complete sequence and bioinformatics analysis.3. The mRNA expression level of the novel spliced variant, Which was designated as NgalR-3 was detected in different esophageal carcinoma cells, esophageal carcinoma, adjacent normal mucosa tissues and normal tissues.4. The potential translated products of alternatively spliced NgalR-2 and NgalR-3 mRNA were evaluated by western blottirig. Subsequently, Immtmofluorescence cell staining analysis and co-immunoprecipitation experiments were performed to see whether we could detect any evidence of co-localization and interaction between NgalRs and NGAL protein.5. Mammalian stable expression vector, which express proteins containing the N-terminal c-Myc epitope tag to finally obtain Myc-NgalR-2 and Myc-NgalR-3, were constructed and the plasmids were transfected into HeLa cells. The cell clones expressed NgalR proteins were obtained by G418 screening.Results and conclusions:1. RT-PCR analysis results demonstrated that both NgalR-1/2, the receptors of NGAL, were expressed in esophageal carcinoma cells, esophageal carcinoma and adjacent normal mucosa tissues, which also expressed NGAL according to our previously study. This result implied that NGAL mediated iron delivery process might present in the esophageal inflammatory epithelium and carcinoma tissues.2. cDNA cloning of human NgalRs variant from esophageal carcinoma EC18 cells and subsequent sequence analysis identified a new NgalR isofrom designated as NgalR-3 that result from alternative splicing. This new isoform encodes a predicted 207 amino acid protein which presents identical amino sequences in the N-terminal 175 amino acids compare with NgalR-2 and contain a unique 32-aa (amino acids 176-207) carboxyl terminus.3. RT-PCR analysis results indicates that this new variant was widely expressed in human normal tissues and esophageal carcinoma cell lines, and the ralative expressed level of NgalR isoforms depended on the origin of tissues and cells. Interestingly, it is noteworthy that NgalR-3 exrpession was up-regulated in 70% cases of esophageal carcinoma in comparison with the adjacent normal epithelium, whereas only in 55% specimen NgalR-1/2 expression was up-regulated. This result could imply that this new NgalR-3 variant might play a more important role in esophageal carcinoma.4. Structure prediction analysis indicates that this novel spliced variant might localize in membrane similar to other NgalR isoforms and was expected to retain its bioactivity. In agreement with this prediction, as expressed in a heterologous system (COS-7 cells), this novel isoform confered the expected function of co-localization with NGAL protein mainly at cell membrane. Further research demonstrated that a physical interaction between NGAL and NgalR-3 could occur in mammalian cells. All of these results suggested that NgalR-3 acted as a potential NGAL receptor in esophageal epithelial cells.5. The HeLa cell lines stable expressed NgalR-2 and NgalR-3 proteins were obtained by G418 screening, which will enable further studies of NGAL mediated iron delivery process, as well as permit studies of the rolls of different NgalR alternative splicing variant.