Mouse retinal pigmented epithelial cell lines retain their phenotypic characteristics after transfection with human papilloma virus: a new tool to further the study of RPE biology

Abstract

Development of immortalized mouse retinal pigmented epithelial cell (RPE) lines that retain many of their in vivo phenotypic characteristics, would aid in studies of ocular diseases including age related macular degeneration (AMD). RPE cells were isolated from 18-month-old (estrogen receptor knockout) ERKOalpha and ERKObeta mice and their C57Bl/6 wildtype littermates. RPE65 and cellular retinaldehyde binding protein (CRALBP) expression, in vivo markers of RPE cells, were detected by real-time RT-PCR and western analysis. We confirmed the presence of epithelial cell markers, ZO1, cytokeratin 8 and 18 by immunofluorescence staining. In addition, we confirmed the distribution of actin filaments and the expression of ezrin. To develop cell lines, RPE cells were isolated, propagated and immortalized using human papilloma virus (HPV) 16 (E6/E7). RPE-specific markers and morphology were assessed before and after immortalization. In wildtype littermate controls, there was no evidence of any alterations in the parameters that we examined including MMP-2, TIMP-2, collagen type IV, and estrogen receptor (ER)alpha and ERbeta protein expression and ER copy number ratio. Therefore, immortalized mouse RPE cell lines that retain their in vivo phenotype can be isolated from either pharmacologically or genetically manipulated mice, and may be used to study RPE cell biology.

Figure 1. Mouse RPE sheets are viable and isolated cells retain their cobblestone appearance

A. RPE sheets are viable after isolation. Live cells fluoresce bright green, whereas dead cells fluoresce red. Comparison of immortalized mouse RPE cells (B) and human RPE cells (C). Mouse RPE cells retain their cobblestone appearance in vitro and have a similar appearance to human RPE cells, D. HPV protein was expressed by all cell lines tested as determined by Western analysis.

Figure 2. Growth Curve of representative RPE cell lines

Cells were plated in 10% containing medium and counted following a 24 hour attachment. Wells were counted on days noted on the x axis of the graph. Shown are two representative RPE cell lines (□) (●)

Figure 3. RPE65 and CRALPB, markers of RPE cells, are expressed in mouse immortalized cells

Western analysis for RPE65 and CRALPB was performed on immortalized, passage 20 RPE cell lysates as described in methods. Lanes 1 and 2: wt RPE cells no injury; lane 3: ERKOβ no injury, lane 4; ERKOα no injury, lane 5: wt cells isolated from eyes injured in vivo, lane 6: ERKOα isolated from eyes injured in vivo, lane 7: ERKOβ isolated from eyes injured in vivo, and lane 8 wt cells non transfected no injury. The same blot was stripped and reprobed with an antibody to actin to ensure equal loading of each lane.

Figure 4. Immunofluorescent localization of ZO1 (A) and Cytokeratin 8 (B) and Actin (C) are expressed in the pattern characteristic for epithelial cells, on mouse RPE cells in vitro

A: ZO1 positive bands are localized between RPE cells. B and C: Cytokeratin and actin filaments localize to the periphery of the cell. Magnification x 40.

Figure 5. Ezrin expression

A and B are XY projections of the entire 3D stack. C is a XZ reconstruction of a 10-voxel thick section (Slidebook 4.2) Light Blue DAPI (DNA), red, ezrin. Bars: AB 20μm C., μm 10. D. Western blot analysis of three representative cell lines (#1 and #3; wildtype cell lines, #2; ERKOβ cell line) revealed the presence of ezrin protein which increased over time.

Figure 5. Ezrin expression

A and B are XY projections of the entire 3D stack. C is a XZ reconstruction of a 10-voxel thick section (Slidebook 4.2) Light Blue DAPI (DNA), red, ezrin. Bars: AB 20μm C., μm 10. D. Western blot analysis of three representative cell lines (#1 and #3; wildtype cell lines, #2; ERKOβ cell line) revealed the presence of ezrin protein which increased over time.

Figure 6. Junctions are present in immortalized mouse RPE cells

Mouse RPE cell lines were grown on coverslips for 10 days and fixed for EM as described in methods. Representative photograph of RPE cell. Upper insets depict a single “kiss” tight junction. Scale bar 2μM

Figure 7. ERα(A) and ERβ(B) protein expression in mouse RPE cells is not altered by immortalization

Western analysis of RPE cell lysates as described in methods. A. Lanes 1–3 represent three individual cell lines. B. Lanes 1–4 represent four individual cell lines. Rec; recombinant protein. Arrow indicates band of interest, 65kDa for ERα and 57kDa for ERβ.