CD46 is an immune gene which covers a diverse range of functions11. This particular protein can integrate several danger signals as well as it can coordinate several immunological interactions12,13 This gene can regulate the immunological homeostasis by regulating the formation of membrane attack complex and thus it can prevent the destruction of self-cells.11,12 Other than these important functions, the role CD46 in regulating the epithelial cell integrity and repair had been discussed well in a recent research article. It states that activation of CD46 resulted in the increased cell proliferation and wound healing in intestinal epithelial cells10. Hence the normal functional role of CD46 on intestinal epithelial cells is homeostatic by the barrier repair after the entry of a pathogen. In the current study we have tried to find out the homeostatic role of cTecrem; a CD46 like protein present in teleost fishes, using carp fin epithelial cell lines. The flow cytometry analysis carried out using both cell aggregates and single cells showed a higher percentage of cTecrem expression when the analysis was done using cell aggregates. This suggested that cTecrem expression may be related to the cell aggregation in some way. So an ELISA was performed using KF-1 cells. In the assay, the cells were passaged and were seeded on to the ELISA plate and the corresponding primary and secondary antibodies were added on to that. The A405 was taken at different points of time. In this assay, an increase in the cTecrem expression over time was observed. Cells were further analyzed under the microscope and observed that the expression of cTecrem is getting higher as the cells got well adhered to the plate. A cell adhesion assay gave further confirmation cTecrem has a role in KF-1 cell adhesion. The number of cells which got adhered to the plate were much higher in the wells coated with Anti-cTecrem antibodies compared to its isotypes. As the antibodies which were coated on the plate could have attracted the cells because of its structure, the increased cell adhesion is due to the stimulation cTecrem is not affirmed. To confirm the role of cTecrem in the increased cell adhesion, a second cell adhesion assay was performed. Since the cells were pre-incubated with Anti-cTecrem antibodies and its isotypes in this assay, the increased cell adhesion observed in the wells where the cells pre-incubated with Anti-cTecrem MAb was added could be due to the effect of Tecrem. It has been proven in CD46 that when the CD46 gene is activated it causes changes in the formation and constellation of adherence and tight junctions via SPAK and E-cadherine10. Since most of the functions of CD46 and cTecrem are similar this could be true for this wound healing function too. So a wound healing assay was performed using KF-1 cells. The results clearly showed a significant increase in the wound healing of KF-1 cells when they were treated with Anti-cTecrem antibodies mainly Anti-cTecrem mAb and Anti-SCR 1,2 pAb. The adhesion of KF-1 cells to the plate was much higher when it was treated with Anti-cTecrem mAb and Anti-SCR 1,2 pAb in the experiments performed. These data in account with the epitope mapping results of Anti-cTecrem mAb shows that the functional domains of cTecrem could be SCR 1 and SCR 2.
The immunohistostaining results showed an increased fluorescence in the wells were Anti-cTecrem antibodies were added compared to the wells without Anti-cTecrem Antibodies. And also the well-defined cell tight junction margins were also observed in the cells incubated with Anti-cTecrem antibodies compared to the controls. This shows that the integrity of the epithelial cells is increased when the cTecrem is activated/stimulated. At this stage of our understanding, the cTecrem would have played a role in the regulation of TJ protein formation in the epithelial cells which would have resulted in the increased cell adhesion and proliferation as we showed in the wound healing assay. In the case of mammalian ZO1 protein, there are many reports which suggest that ZO1 protein is produced in a minimal amount when the cell proliferate24,26,29. ZO1 protein in mammals is found to have a role in tumor suppression as well 25,26,27. But the amount of ZO1 protein is found to increase after the cell forms a defined monolayer and start to differentiate29. The upregulation of the ZO1 protein happens after the formation of the rigid monolayer24,27,28. cTecrem might be influencing this upregulation which might have caused in the increased fluorescence in the cTecrem activated CFS cells. A study done in the rainbow trout gill epithelial cells also shows an increase in the ZO1 transcript abundance in the gill cells at the time of monolayer formation30. This study suggests that the increase in the ZO1 mRNA indicates the role of ZO1 protein in establishing the cell to cell contact as well as the gill epithelium integrity. The cTecrem could be playing a role in the upregulation and the downregulation of the ZO1 proteins. Hence the activation of cTecrem would have resulted in the kickstart of epithelial cell proliferation as well adhesion. The ZONAB (ZO1 associated nucleic acid binding protein) protein identified in several organisms is found to have a role in the increase of cell proliferation. This protein shares homology with zebrafish as well. ZONAB protein is found to be up-regulated at the time of cell proliferation and is found to be down-regulated after the formation of the monolayer. Since zebra fish shares a homology with some of the carp proteins this protein can be present in carp as well though we have not investigated it yet. The immunohistostaining results suggest that the activation of cTecrem resulted in the hypersecretion of the ZO1 protein. Though it looks contradictory to the results in mammalian cells, it could be because of the changes in the time point where the analysis was done. The variation in the levels of ZO1 mRNA at different time points in the Zebra fish flask-cultured epithelial cells is in agreement with our present study30.
The assay done for analyzing the change in the compactness of the cell monolayer up on the activation of cTecrem also gave similar results with the immunohistostaining. Both CFS and KF-1 cells were grown for 3 days with and without adding anti-cTecrem antibodies. The morphology of several flask-cultured vertebrate epithelial cells has two distinct stages of development as per the previous reports available. The first stage is the attainment of confluence during the first 72 h which includes the cell-cell contact and the polarity changes. In this stage, the proliferation stops due to the contact inhibition31. And the second stage is the establishment of resistive properties and in this stage, the borders of the cell monolayer will become clearly visible31.This is why we chose to start our analysis from the third day of culture. On the third, fourth and seventh-day of cell seeding, the area of 50 cells from each sample and control group was calculated using ImageJ software. The graph plotted with the average area of each group showed a decrease in the cell area of the cells activated with cTecrem antibodies. The decrease in the cell area shows the increase in the monolayer compactness. Comparing this data with the immunohistochemistry data suggests that, the tight junction proteins were hyper secreted upon the activation of cTecrem. This might be the reason for the increased compactness of the CFS and KF-1 monolayers when they are activated with cTecrem antibodies.